KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7–3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model. KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Here the authors use cryoEM to determine 20 structures of KIF14 constructs bound to microtubules in the presence of different nucleotide analogues and provide the structural basis for a coordinated chemo-mechanical kinesin translocation model. Its overexpression is associated with tumor progression and resistance to anticancer drugs in several cancers for which it is considered an oncogene9. Mutations in KIF14 are also associated with neural and kidney development defects11. However, despite the importance of KIF14, its action mechanism as a kinesin motor is still largely unknown.KIF14 is a microtubule-based motor protein with essential roles during cell division12. Kinesin-3s in general work as microtubule plus-end-directed motors with the ability to make long processive runs when forming dimers13. KIF14 is also reported to be a microtubule plus-end-directed motile kinesin and to protect microtubules against depolymerization14. As all kinesins, KIF14 possesses a highly conserved catalytic motor or head domain that contains nucleotide and microtubule binding sites, and where ATP hydrolysis is coupled to the generation of mechanical work. The KIF14 molecule is also similar to other microtubule plus-end-directed motile kinesins in having two motor domain joined by a coiled coil dimerization domain located C-terminal to the motor domain and a ~13 residues long region, the neck-linker, connecting the motor and coiled coil domains15. KIF14 also contains an N-terminal extension with a protein-regulating cytokinesis 1 binding domain14.KIF14 belongs to the kinesin-3 family of motor proteins. Other members of this family include KIF1A and CeUNC-104Although much is known regarding the mechanism of action of motile kinesins, and in particular of its founding member kinesin-1, most structural information of kinesin-microtubule complexes is still of limited resolution. As a consequence, it is still not fully clear how conformational changes are coupled to distinct steps of the ATP hydrolysis cycle or how the two heads of a kinesin dimer may coordinate their activities.Here we report the near-atomic resolution structures of twenty KIF14-microtubule complexes corresponding to five distinct KIF14 constructs in four nucleotide conditions mimicking four key steps of the ATP hydrolysis cycle. We also implemented an analysis procedure to separate coexisting conformations of the kinesin motor bound to the microtubule helical assembly. With this procedure, we determined the structure of the two motor domains of KIF14 dimers bound to the microtubule (two-heads-bound state) at high resolution. The data reveal how microtubule binding alters the structure of the KIF14 motor domain, how changes in nucleotide species bound to the catalytic site are coupled to motility-related conformational changes and how the two motor domains in a KIF14 dimer coordinate their activities. Our structural data provide a comprehensive molecular understanding of how kinesin ATPase cycle is coordinated with plus-end-directed movement along microtubules.17. Here, we take KIF14 Ile-736 as the first neck-linker residue, which is analogous to the definition used in refs. 18. KIF14 Ile-736 aligns with kinesin-1 KIF5B Ile-325. The limits of KIF14 CC1 (Ile-751 to Ala-764) were taken as the residues with a higher than 0.99 COILS19 coiled coil score. The longer K772 construct includes a full neck-linker and the first coiled coil domain CC1. Construct K755 includes the neck-linker and part of the first coiled coil heptad repeat of the CC1 domain. Construct K748 includes 13 neck-linker residues and no part of the CC1 domain. Construct K743 includes 8 neck-linker residues. Construct K735 comprises the core motor domain and no neck-linker residues. Construct K772 with a full CC1 domain migrates faster than the shorter constructs in a size-exclusion chromatography column  to mimic the ADP-Pi transition state. The four nucleotide conditions will be denoted through the text as ADP, Apo, ANP, and AAF, respectively.To elucidate conformational changes related to the microtubule-bound KIF14 motor domain ATPase cycle, and their possible modulation by the neck-linker or a partner motor domain, we determined the cryo-EM structures of microtubule (MT) complexes of five distinct KIF14 constructs in four nucleotide conditions. Full-length mouse KIF14 is 1674 amino acids long and the core motor domain resides between residues Asn-391 and Leu-735 Fig. . The fivumn Fig. . The fas14.Combining the different KIF14 constructs and nucleotide conditions, we first calculated eighteen 3D cryo-EM maps using a helical reconstruction method where all the asymmetric subunits in the microtubule helical assembly get averaged. Twelve of these cryo-EM maps revealed one of the two alternative conformations of the KIF14 motor domain while the other six revealed a mixture of conformations averaged. To separate the mixed conformations, we applied to all eighteen datasets a procedure  are not resolved, and thus presumed disordered. On the other hand, all the KIF14 microtubule-bound cryo-EM structures show clear densities along the full length of these regions , indicative of the formation of a salt bridge between these residues, are resolved in the kinesin-microtubule complexes ons Fig. . KL9 and27. We also propose that the open nucleotide conformation and the large displacement of KH0 relative to the plus subdomain facilitates the incorporation of ATP into the nucleotide-binding pocket and the exchange of ADP for ATP. This is consistent with molecular dynamics simulations that link movements of KH0, KL5, and SW1 with the incorporation of ATP into the nucleotide-binding pocket28. Also, consistent with this proposal, we find that ATP analogs can readily bind to the open nucleotide-binding conformation  neither analog induces the closed configuration of the motor domain observed with the constructs that include at least 13 neck-linker residues.The motor domain in the MT-K735-ANP complex shows a new configuration where KH0 is at an intermediate position between the open and closed configuration Fig. , but as x also do not fully conform to the closed configuration. The helically averaged cryo-EM maps are best-fitted with an open configuration KIF14 model, but different from the MT-K735 complexes, these maps also include weaker densities corresponding to the closed configuration . Conversely, preventing neck-linker docking impedes full closure of the nucleotide-binding pocket and inhibits the ATPase activity.Consistent with the structural results, we found that the K735 and K743 constructs have reduced microtubule-stimulated ATPase activity relative to the longer KIF14 constructs Fig. . This is2+ ion, and switch-1 Ser-603.The open* conformations MT-K735-ANP/AAF and MT-K743-O-ANP/AAF cryo-EM maps show clear nucleotide-associated densities in the active site indicating that both nucleotide analogs bind to the open* configuration of the motor domain Fig. . In the x or the Mg2+ ion are near the noise level. The weak or absent AlFx densities and the lack of Mg2+ coordination indicate a weakened interaction of the Pi mimic in the active site. This suggests that neck-linker undocking  and the consequent opening of the nucleotide-binding pocket promote Pi release from the active site after ATP hydrolysis.The MT-K735-AAF and MT-K743-AAF maps contain strong densities associated with ADP but densities that could have been attributed to AlFThe structure of the core motor domain of the MT- K755 and MT-K772 complexes in the Apo and ADP states conform to the open configuration, as it is also the case for the complexes with the shorter KIF14 constructs Fig. . Extra dx states, the MT-K755 and MT-K772 helically averaged cryo-EM maps show a density connecting the motor domains along a protofilament .Different from the Apo and ADP states in the AMP-PNP and ADP-AlFent Fig. . This deent Fig. . In addient Fig. . The necThe structures of the leading and trailing heads of the MT-K755-ANP/AAF and MT-K772-ANP/AAF helically averaged cryo-EM maps were separated using the 3D refinement and classification procedure  or pointing sideways Fig. . On the We have solved the near-atomic resolution cryo-EM structures of five distinct KIF14 protein constructs bound to microtubules, in four nucleotide conditions mimicking key steps of the KIF14 ATPase cycle. Our data provides a comprehensive view at the highest resolution available of the mechano-chemical cycle of a motile kinesin and provide the structural basis for a mechano-chemical model for kinesin plus-end translocation Fig. . In the 18. In both cases, the nucleotide-binding pocket becomes more open relative to the tubulin-unbound structure, but there are also significant structural differences between the complexes. In the kinesin-1-tubulin Apo structure, the switch-1 loop is mostly disordered and becomes ordered in the presence of ADP-AlFx18. On the other hand, in all the KIF14-microtubule structures, including the Apo state, this loop is mostly ordered. The relative orientation of the motor domain relative to the bound tubulin is also slightly different , different tubulin structures (curved vs. straight) or the resolution of the cryo-EM structures is not fully clear yet. To address this issue, structural studies of other kinesin-microtubule complexes at similar resolution are needed. Although there are several cryo-EM structures of kinesin microtubule complexes available, only recently it has become possible to achieve the resolution needed (<4 Å in the kinesin part of the map) to fully trace the polypeptide chain, to resolve the positions of side chains, to identify the nucleotide species in the active site, and to fully resolve the conformation of exposed and potentially mobile regions, such as the switch loops and the neck-linker.The microtubule-bound KIF14 open structures share some similarities with the near-atomic resolution crystal structure of kinesin-1 bound to a curved tubulin in the Apo statex states18, but they are not identical. The transition between the open and closed conformations of the KIF14 motor domain can also be described as a clamshell-seesaw type movement between subdomains36. However, a distinction between our model and previous ones is that we recognize that the three moving subdomains form part of the tubulin–kinesin interface and the nucleotide-binding pocket is located between them. This, we propose is the key factor coupling the kinesin ATPase cycle with microtubule binding, as relative movements between the subdomains simultaneously alters the structures of the microtubule-binding interface and the nucleotide-binding pocket  can be approximated as movement between three subdomains we called the plus, minus, and switch subdomains  are insufficient to fully stabilize the docked conformation. In contrast, for the non-motile kinesin-13 KLP10A only four neck-linker residues are sufficient to stabilize a docked conformation and a closed nucleotide-binding pocket when the motor domain is bound to the curved tubulin37. Thus, controlling closure of the nucleotide-binding pocket and entering the hydrolysis competent step appears to be a general regulatory mechanism in the kinesin super-family. Although we cannot rule out small changes in nucleotide affinity, we do not expect them to be a limiting factor at physiological molar concentrations of ATP, similar to the ones used in this study.The capacity of the kinesin motor domain to bind nucleotides, regardless of the position of the neck-linker or whether the nucleotide binding pocket is open or closed is unlikely to be a feature unique to KIF14. A recent structure of another kinesin (kinesin-13), where the nucleotide-binding pocket is prevented from closing by a different mechanism, also show an ATP analog bound in the active site45. The MT-K755-ANP/AAF and MT-K772-ANP/AAF complexes show the leading and trailing heads in two distinct conformations providing structural evidence of coordination between the two motor domains. The ATPase cycles of the two motor domains are kept out of phase as the undocked neck-linker of the leading head keeps the catalytic site in the open configuration inhibiting ATP hydrolysis . Instead, the nucleotide in solution is always found bound, regardless whether it is the leading or the trailing head or whether the active site is open or closed .The longer KIF14 constructs that includes part of the coiled coil dimerization domain, K755 and K772, show at near-atomic resolution what the structures of a kinesin dimer two-heads-bound intermediate looks like. Such intermediate is present in most models of kinesin plus-end-directed processive movement and it is likely to be the dominant state during translocation at physiological ATP concentrationssed Fig. . This is50. The conformation of the neck-linker in the K772 and K755 Apo/ADP structures suggests that the tethered head is positioned sideways and toward the microtubule plus end (the direction of movement) relative to the trailing position seen in the two-head microtubule-bound intermediate  to generate the untagged motors. KIF14 constructs were flash-frozen in small aliquots in liquid nitrogen, and stored at −80 °C freezer.KIF14 constructs were generated as GST-fusion proteins by amplification of the desired constructs from a plasmid containing the corresponding cDNA using polymerase chain reactions. The plasmids were transformed into BL21 (DE3) pLysS cells and were induced with 0.5-mM IPTG at 18 °C overnight. GST-fusion proteins were purified on glutathione resins (Genscript)37.Fifteen-protofilaments-enriched microtubules were prepared from porcine brain tubulin 51. Briefly, reactions were assembled in BRB40-based buffer , 25-mM KCl, 0.25-mg/mL BSA, 1-mM DTT, 0.02% Tween), supplemented with 1-mM ATP, 2 μM of paclitaxel-stabilized microtubules, and 50-nM KIF14 protein constructs. Basal activity of the KIF14 constructs was determined using the same reaction condition with no microtubules added. Reactions were allowed to proceed for 10 min, quenched with perchloric acid and malachite green reagent14. The signal was quantified by the absorbance at 620 nm in a Genios Plus plate reader (Tecan).A malachite green-based phosphate detection assay was used to measure KIF14-mediated ATPase activity2 to ADP or AMP-PNP. Four microliters of a microtubule solution at 2–5-μM tubulin in BRB80 plus 20-μM paclitaxel were layered onto the EM grid and incubated 1 min at room temperature. This microtubule solution also contains either AMP-PNP at 4 mM, ADP at 4 mM, or ADP at 4 mM plus 2-mM AlCl3 and 10-mM KF. During the incubation time, a fraction of the thawed kinesin aliquot was diluted to prepare a 20-μM kinesin solution  containing 20-μM paclitaxel and either of the four nucleotide conditions to be probed: (1) 4-mM ADP, (2) apyrase: 5 × 10−3 units per µl (Apo conditions), (3) 4-mM AMP-PNP, and (4) 4-mM ADP plus 2-mM AlCl3, and 10-mM KF (ADP-AlFx conditions). Then the excess microtubule solution was removed from the grid using a Whatman #1 paper. Four microliters of the kinesin solution was then applied onto the EM grid, transferred to the chamber of a Vitrobot apparatus (FEI-ThermoFisher MA) at 100% humidity where it was incubated for 1 min at room temperature, and blotted for 2.5–3 s with a Whatman #1 and −2-mm offset before plunge-freezing into liquid ethane. Grids were clipped and stored in liquid nitrogen until imaging in a cryo-electron microscope.MT-K735-ANP, MT-K735-Apo, and MT-K772-ANP datasets were collected on untreated carbon/copper grids (Quantifoil R2/2 300 mesh). The grids used for the other 15 datasets were gold grids (UltrAuFoil R2/2 200 mesh) plasma cleaned just before use . Microtubule was polymerized fresh the day of the cryo-EM sample preparation. Kinesin aliquots were thawed on ice just before use. All nucleotides stock solutions used were prepared with an equimolar equivalent of MgCl52 with the image-shift protocol and partial correction for coma induced by beam tilt53. Data collection was mainly performed semi-automatically using 3–5 exposures per 2-μm diameter holes. The exposures were fractionated on 35–50 movie frames. The defocus ranges and cumulated dose are given in Table Data were collected at 300 kV on Titan Krios microscopes equipped with K2 summit detectors  Å−1 and run until no further improvement in resolution was detected. Number of particle images and asymmetric units included in the 3D reconstructions are reported in Table 57 against their respective half-reconstruction and without particle alignment . A moving median over five contiguous particles was applied on each microtubule to assign their final per-particle defocus values. After CTF refinement, further Frealign cycles limiting the refinement data to (1/5) Å−1 were run until no further improvement in resolution was detected. A Frealign 3D classification into two classes was used to improve the signal to noise (S/N) of the 748-ANP dataset. Unmasked–unfiltered–unsharpened Frealign reconstructions were obtained for each half dataset and helical symmetry was imposed in real space to each half map with relion_helical_toolbox. These half maps were merged and corrected for the modulation transfer function of the detector by relion_postprocess.Images of 15R microtubules were processed using a helical-single-particle 3D analysis workflow58 on 12-pixel size boxes to obtain initial local resolution estimates. The merged map was locally filtered by blocfilt58 using blocres local resolution estimates and then used for local low-pass filtration and local sharpening in localdeblur59 with resolution search up to 25 Å. The localdeblur program converged to a filtration appropriate for the tubulin part of the map but over-sharpened for the kinesin part. The maps at every localdeblur cycle were saved and the maps with optimal filtration for the kinesin part area were selected. Finally, helical symmetry was imposed to the locally filtered maps.To obtain a final locally filtered and locally sharpened map post-processing of the pair of unfiltered and unsharpened half maps was performed as follows. One of the two unfiltered half-map was low-pass-filtered to 15 Å and the minimal threshold value that does not show noise around the helical segment was used to generate a mask with relion mask create on 85% of the helical segment on its helical axis . This soft mask was used in blocres49, HASRC can separate two-bound kinesin dimer complex structures in fully decorated microtubules at near-atomic resolution. HASRC was implemented in Relion 3.1 adapting ideas and methods previously used to separate and classify the subunits contained in symmetrical multi-subunit assemblies61. These ideas have also been used recently to refine the structure of microtubule protofilaments62. We implemented specific subunit location refinement and classification steps in HASRC that were essential to separate coexisting kinesin motor domain conformations at high resolution.The helical-single-particle 3D reconstruction procedure generates density maps where all the asymmetric units in the helical assembly (one tubulin heterodimer with one-bound kinesin motor domain) are averaged. To independently determine the structure of the two motor domains of kinesin dimers bound to the microtubule (two-heads-bound), we implemented a procedure (HASRC) to isolate and classify individual subunits in a helical assembly. Different from a previous methodIn addition to the ability to separate coexisting subunit structures, HASRC allowed to account for local lattice distortions, which resulted in up to 0.4-Å resolution improvement over the helically averaged maps. Thus, to produce maps at the highest possible resolution, we applied HASRC to all datasets, dimers in the two-heads-bound states as well as monomers and dimers in the one-head-bound states.Relion helical refinement. The two independent Frealign helical refined half datasets were subjected to a single helical autorefinement in Relion 3.1 where each dataset was assigned to a distinct half-set and using as priors the Euler angle values determined in the helical-single-particle 3D reconstruction .full) was generated from a KIF14 dimer model bound to two tubulin dimers while the other mask (maskkinesin) was generated with only the kinesin coordinates. The helical dataset alignment file was symmetry expanded using the 15R microtubule symmetry of the dataset. Partial signal subtraction was then performed using maskfull to retain the signal within that mask. During this procedure, images were re-centered on the projections of 3D coordinates of the center of mass of maskfull (CM) using a box size as indicated in Supplementary Table Asymmetric refinement with partial signal subtraction. An atomic model of a KIF14 dimer two-heads-bound state was used to generate two soft masks . Two of the resulting classes contained two well-resolved kinesin motor domains while the others had absent or not well-resolved kinesin densities . To avoid potential artefacts introduced by the signal subtraction procedure, final 3D reconstructions were obtained using relion_reconstruct on the original image-particles without signal subtraction. Map filtration was then performed the same way as with the helically averaged maps, without symmetry imposition.For each of the four KIF14 dimer two-heads-bound states datasets , the following procedure was used  were found  the same procedure as described above was employed with the following modifications  were selected. Fourth, the Rosetta-refined models were further refined against the cryo-EM density maps using Phenix real space refinement tools67. Fifth, the Phenix-refined models were edited using Coot68. Several iterations of Phenix real space refinement and Coot editing were performed to reach the final atomic models.Atomic models of the cryo-EM density maps were built as follow. First, atomic models for each protein chains were generated from their amino-acid sequence by homology modeling using Modeller64 or VMD69. Movies were made with VMD69 and R70.Atomic models and cryo-EM map figures were prepared with UCSF-Chimera71 that test the convergence of independently fitted atomic model to the cryo-EM densities. Each coordinate model was refined against the corresponding cryo-EM density map using Rosetta66. Two hundred independent models were generated, 100 using the refine-torsion protocol and 100 using the refine-cartesian protocol. The ten best models produced by each protocol (best fit to density and best molprobity scores) were then subjected to real-space refinement in Phenix67. Precision was expressed as the root mean square deviation between equivalent Cα carbon coordinates of the 20 resulting models (Supplementary Fig. To estimate the precision of the model atomic coordinates fitted into the cryo-EM density maps, we used the method proposed by Herzik et al.64. When comparing the density of equivalent regions in different maps and atomic models, the maps and models were first aligned either to their corresponding β-tubulin (open vs. closed densities, Supplementary Fig. The relative intensity of different regions of the cryo-EM maps (Supplementary Figs. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Reporting Summary


Pauling described metallic bonds using resonance. The maximum probability domains in the Kronig–Penney model can show a picture of it. When the walls are opaque (and the band gap is large) the maximum probability domain for an electron pair essentially corresponds to the region between the walls: the electron pairs are localized within two consecutive walls. However, when the walls become transparent (and the band gaps closes), the maximum probability domain can be moved through the system without a significant loss in probability. Nature of the chemical bond and the structure of molecules and crystals [Years ago, I met a famous physicist (whose name should not be divulged here). He said that Pauling had many good ideas, but that he had been wrong about one thing: metals. Of course, there are many aspects related to metals, some of which go beyond their electronic structure; these are of interest in the present paper. So, how did Pauling see metals? In his crystals , he descInstead of performing valence bond calculations for metals, this paper searches for electron pairs in the very simple model of Kronig and Penney . It is tWe search for a domains, maximizing the probability to find a pair of electrons in themWe find that in the insulator regime, these domains are well-defined in space, while in the metallic regime a translation produces an equivalent domain.b and height L. In the model of Kronig and Penney , one deaL increases, bands are formed. A continuum of levels appears between The energy levels, P, is maximal. We thus occupy each of the energy levels up to N arises because the electrons are not distinguishable. A numerically efficient way to compute this was given by E. Cancès [P is In order to connect with bonds, we determine the interval  maximal ,4. As we. Cancès . The maxThe corresponding domain (the maximum probability domain) is All computations were conducted with Mathematica .L, become very large. However, in computing the probability, L increases. Fortunately, the convergence with L is fast, as can be seen in L and L chosen below.In order to study crystals, we should let the number of cells, Equation , becomesP changes with the opacity, P, P as a function of P increases with Let us now turn our attention to the domain maximizing Equation . Figure We are treating the Kronig–Penney one-dimensional model, cf. Equation , when itThe calculations have revealed another feature — namely, that, as Equation , is not Of course, the picture in three dimensions, 3D, is different. Furthermore, atoms have cores, and the Pauli principle produces spherical “walls” around the nuclei. Nevertheless, we can expect a similar behavior, in the sense that cores can be avoided in 3D, and “channels” exist through which displacement hardly changes the probability to find an electron pair in them. This is supported by calculations with the electron localization function , ELF — slic β-Sn .The phenomenon of deforming the optimal domain, g., Ref. ). Such a


DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging.  OGDH gene leading to deficient 2-oxoglutarate dehydrogenase  was reported in individuals with a neurological disorder resembling mitochondrial disease . Fur. FurOGDHpatients . The DHT pathway . Genetic 204750) ,10,11, C 204750) ,13,14 an 204750) .GCDH gene encoding the mitochondrial protein glutaryl-CoA dehydrogenase  located downstream of E1a in the L-lysine degradation pathway , a metabolic disorder that is caused by mutations in the  pathway  15]..GCDH genOGDH, OGDHL and DHTKD1 variants and their impact on E1o, E1o-like and E1a protein structure and function.Despite the reported genetic findings, no pathophysiologic mechanism has been reported for the disease-associated OGDH and LIAS (encoding lipoic acid synthase) were identified that are involved in the mechanism of regulation in hypoxia-inducible transcription factors (HIFs) under aerobic conditions . E. EMsKGD nterface , top ,88MsKGD n MsKGD) C. The ε-n MsKGD) C [35]. In MsKGD) D. Earlietida E1b , where H2+-binding site, including peptides 485–505, 506–518 and 646–664 in E1a displayed a lower relative deuterium incorporation, suggesting less accessibility to the deuterium on interaction with E2o. However, the peptide comprising residues 407–415 in the same region of E1o displayed modestly increased deuterium uptake  si si85] sim uptake ,85. In Fm uptake . These H37, Lys148 and Lys188), in the ThDP-Mg2+-binding region (Lys300 and Lys450), and in the C-terminal region  , from the linker region , and from the inner core domain   experiments were carried out by using two cross-linkers: 1,1′-carbonyldiimidazole, a zero-length cross-linker  and disuccinimidyl dibutyric urea . The majority of cross-linked residues identified in E1a are located in the N-terminal region Lys852/854) . Lysine 2/854 . Ltructure ,91,92,9325-919 [MsKGD  as the search template. The E2o structure was built by using multiple templates with I-TASSER . In the second stage, the incompatible intra-protein (or intra-component) cross-links were filtered by using the Xlink Analyzer [To gain a deeper understanding of the human E1o-E2o and E1a-E2o interactions in binary sub-complexes, cross-linked guided modeling was performed by using web servers and structural prediction algorithms reported in the literature ,102,103.25-919 . The E1oAnalyzer  in visuaAnalyzer  by applyAnalyzer . The comAnalyzer . In the Analyzer  and the 2+-binding site to provide the catalytic mechanism for succinyl/glutaryl transfer between the E1o/E1a and E2 components. On the other hand, three regions of the E2o core domain with α-helix secondary structure comprising residues 191–208 from the linker region and residues 370–386 from the E2o core are located proximal to E1o, while the region comprising residues 273–289 from the E2o core domain points away from the E1o  and E1a (residues 24–47) from H/D exchange in accord with HDX-MS findings where the N-terminal region of E1o and E1a displayed significant deuterium uptake decrease on interaction with E2o. Also, the lipoyllysyl-arm of the E2o comprising residues 68–128 of the lipoyl domain and residues 129–226 of the linker region preceding the catalytic domain, is swinging around the E1o/E1a ThDP-Mg the E1o B, Left.  the E1o B, Right.E. coli pyruvate dehydrogenase complex (PDHc) and OGDHc were reported three decades ago [E. coli PDHc and OGDHc was reinvestigated by using cryo-electron tomography and it was demonstrated that the E1 and E3 components in these complexes are flexibly tethered ~11 nm away from the E2 core with no significant variations in the E2 core dimensions [E. coli E2o core [E. coli E2p core [A. vinelandii E2p core [45-919-E2o68-453 sub-complex of 2.45 MDa was derived from small-angle X-ray scattering (SAXS) experiments, suggesting a stoichiometry of one E1a dimer per one E2o trimer for their binding [A thorough identification of protein-protein interactions in the family of the 2-oxo acid dehydrogenase complexes remains a challenge. Early studies of the architecture of the 2-oxo acid dehydrogenase complexes by analysis of electron cryo-microscopy images of the ades ago . The impades ago . Some 15mensions . Recentlmensions . The EM mensions . There wmensions . The humE2o core , the E. E2p core  and for E2p core , all obtE2p core . A molec binding , similar binding .Bacillus stearothermophilus [tE2p) core from Saccharomyces cerevisiae (S. cerevisiae) which lacks the lipoyl domain and the E1-binding domain [S. cerevisiae tE2p core generated at 25 Ǻ resolution revealed that the E2p catalytic domains are arranged in cone-shaped trimers at each of the 20 vertices of the dodecahedron structure which are interconnected by 30 bridges [S. cerevisiae E2p core that is due to conformational changes in the trimers and in the bridges that connect adjacent trimers suggesting a flexible “breathing” core, which may be important for multienzyme complex function, such as channeling of intermediates between the active centers of the individual components [S. cerevisiae E2p core in sub-complex with E1p’s generated by cryo-EM at ~30 Ǻ resolution suggested that the length of the E2p inner linkers that anchors the E1p tetramers from the outer shell to the E2p core also may vary from ~50–75 Ǻ depending on the occupancy of the outer shell by E1p’s, contributing to the overall dynamics of the S. cereviciae PDHc [tE2p core (devoid of lipoyl domains and E1p-binding domains) at 8.8 Ǻ and 3.1 Ǻ resolution that also enabled visualization of the secondary structure elements within the tE2p core, including the α helices and β sheets [S. cerevisiae E2p core, exhibited variation in particle size suggesting its flexibility [In contrast to 2-oxoglutarate dehydrogenase complexes, cryo-EM studies of the pyruvate dehydrogenase complexes (PDHc’s) from Gram-positive bacteria such as mophilus , yeast [mophilus ,109,110,mophilus ,112, andmophilus ,79,80 reg domain ,109. The bridges ,109. Theβ sheets ,79. The xibility .In eukaryotes, the complexity of the E2p core is increased, being formed by the E2p and E3-binding protein (E3BP) components, which assemble into a 60-mer central core with icosahedral symmetry . Two altThe 2-oxoacid dehydrogenase complexes are macromolecular assemblies functioning in different metabolic pathways. These assemblies provide high catalytic efficiency and high-level of regulation to the metabolic pathways in the cell and the regulation of gene expression in the nucleus. Recent experimental findings in vitro and in vivo have identified the formation of hybrid complexes between the OGDHc and E1a, suggesting functional crosstalk between the two distinct metabolic pathways and an additional layer of regulation that needs to be thoroughly established. The direct association of the OGDHc and/or OADHc with known histone acetyltransferase KAT2 and histone H3/H4 has recently been discovered, suggesting a functional crosstalk between the KAT2 and OGDHc/OADHc in nuclei for the purpose of posttranslational modification of histones by succinylation/glutarylation and regulation of gene expression, tumor cell proliferation, and tumor formation ,32. Howe


Amorphophallus konjac flour  and to evaluate their nutritional and physicochemical properties. Proteins, lipids, carbohydrates, moisture, ash content, fibers, resistant starch, firmness, specific volume, and color were evaluated using official methods. Protein varied from 2.95% to 4.94%, the energy value from 347.93 to 133.55 kcal/100 g, dietary fiber from 8.19 to 17.90%, and resistant starch from 0.67% to 0.75% on wet basis. The addition of konjac flour positively influenced the specific volume. Higher concentrations of konjac flour in the formulations led to lower calories of the bread due to the significant addition of water to the dough. The bread samples with konjac showed high fiber content due to the composition of the flour. They had lower levels of carbohydrates, which can positively influence the glycemic index. Konjac flour provided dough mold, growth, and better texture for gluten-free bread. The best formulations were prepared in concentrations up to 37.5% konjac. The 50% konjac bread showed slightly reduced specific volume and pale color.The demand for gluten-free products is rising, but their production with similar quality as their gluten counterparts is challenging. This study aimed to develop gluten-free bread samples using different concentrations of  Due to the growing trend in consuming gluten-free products, the food industry has sought to expand and diversify its production to meet this increasing demand . The gluAmong the gluten-free foods that make up the market’s largest share are bakery products , bread bAmorphophalluskonjac . It is used in Chinese medicine for detoxification, cancer suppression, stasis of blood, treatment of asthma, cough, hernia, breast pain, and hematological and skin disorders [Foste et al.  claim thisorders . Due to isorders .Amorphophallus konjac was not broadly studied. The flour from Amorphophallus konjac is considered a functional ingredient [Amorphophallus konjac flour in concentrations of 0.25%, 0.50%, and 0.75% as a thickener in gluten-free bread, and Moore et al. [As a food additive, glucomannan was tested in bakery products, drinks, bread, and pasta ,21, but gredient  containigredient ,24,25. Tgredient . Until ngredient  used Amoe et al.  used konAmorphophallus konjac flour and its potential benefits to individuals suffering from GRD, this study aimed to develop gluten-free bread with Amorphophallus konjac flour and to evaluate the nutritional and physicochemical properties of the formulations with different concentrations of the flour. Considering the potential application of This experimental study took place in the Dietetic Laboratory from the University of Brasilia (Brazil) for bread production. For chemical composition analysis, color, texture, and specific volume, research was conducted in the Food Analysis laboratory (UnB), EMBRAPA, and ITAL (Food Technology Institute). All samples were developed and analyzed in triplicate.Amorphophallus konjac flour was purchased from a Brazilian pharmaceutical company  in three different lots  imported from China in packages of 1000 g each. These three flours were mixed in the exact amounts to prepare the formulations. The used flours were composed of 70% glucomannan konjac, according to the labels. They were previously microbiologically tested  and approved by the Brazilian sanitary legislation. Amorphophallus konjac flour was added in the proportion of 12.5%, 25%, 37.5%, and 50% of the flour quantity. According to the literature, konjac flour presents almost 80% of fibers and only 8% of starch, making it unfeasible to use it above the maximum percentage applied in this study. Preliminary tests were performed with percentages superior to 50%, and bread samples presented a strong odor and “taste of fish”. For the samples added of konjac flour, the amount of sucrose, salt, soy oil, and eggs was the same as the control sample (considering the weight of the control flour basis). The amount of water was adjusted due to konjac flour’s fiber content that can absorb 200 times their weight in water [The control gluten-free bread (GFB) was composed of potato starch (30%) and rice flour (70%) as flour basis, added sucrose (12 g/100 g of control flour basis); salt (3 g/100 g of control flour basis); water (34.5 g/100 g of control flour basis); soy oil (16.5 g/100 g of control flour basis); whole egg (29.5 g/100 g of control flour basis); and yeast (1.5 g/100 g of control flour basis). This formulation was adjusted from the bread formulation studied by Aguiar , and no ®-Brazil, at 180 °C to bake all the samples. On the day of the analysis, samples were prepared and baked. After weighing ingredients, the yeast was pre-activated in sucrose and warm water (38 °C) for 10 min. Separately, rice flour, potato starch, konjac flour, and salt were mixed. Eggs, water, and oil were added to the dry ingredients. Finally, activated fresh biological yeast was added, and the dough was homogenized. This dough was kneaded and rested for 50 min, and then, it went through a second kneading and modeling (40 g spheres). We used a preheated gas oven BrastempThe cooking factor for each sample of bread was determined using the formula proposed by Araujo et al. .(1)CookMoreover, we calculated the weight loss percentage  after baThe Adolfo Lutz Institute’s analytical standards  were use0, a0 and b0 are the coordinates obtained for the control sample.The evaluation of the color of the crust, crumb, and bottom of the bread samples was carried out in a spectrophotometer ColorQuestXE . We obtained from the Hunter system the values for the coordinates L* (measurable in terms of white to black intensity), a* (measurable in terms of intensity from red to green), and b* (measurable in terms of yellow and blue intensity). It was possible to obtain hue angle h* (Equation (3)), color saturation or chroma C* (Equation (4)), and color difference ΔE (Equation (5)) ,36,37,38Measurement of Bread Firmness by Universal Testing Machine [The texture profile (TPA) analysis of the bread samples was performed using the method 74-09.01— Machine . The equ3) and mass (g) of each sample. Specific volume was measured by the rapeseed displacement method  through The total energy value was reached with the macronutrients from the proximate composition analysis and the Atwater factors multiplying fats by 9 kcal/g, proteins by 4 kcal/g, and carbohydrates by 4 kcal/g . As mentp < 0.05 considered significant. Statistical analyses were performed using software SPSS-IBM  All analyses were conducted in triplicate.The results were subjected to one-way ANOVA followed by Tukey’s post hoc test, with the level of p = 0.008). Lipids reduced 26.1% when comparing the control bread and the 25% konjac, and 51.3% compared to the 50% konjac. There was a reduction of 38.1% between the control sample and the 50% konjac sample for carbohydrates. The 50% konjac bread presented 34.5 more times fiber than the control bread for dietary fibers. The total energy value (TEV) decreased by 61.6% between control and 50% konjac samples.p < 0.05) was obtained for SV from the control and all konjac samples, with a higher volume with the increase of konjac. However, only 12.5% bread is different from other formulations, demonstrating that the increase in konjac above 25% did not affect the volume. Konjac flour provided more significant bread expansion, contributing to its texture. It was impossible to measure control bread’s firmness in the same conditions as the other bread samples. Control bread presented a very hard texture after baking since it was only prepared with potato starch and rice flour and no additives to improve texture. The cylindrical probe did not penetrate the sample because of its hardness; therefore, the equipment did not provide reading parameters.The specific volume (SV) is a measure to verify the dough’s ability to expand and retain the gas during baking . Table 3The average values of Chroma-C*, hue angle-h*, and color difference for the different samples are in p < 0.05) from the others. It indicates that as the konjac flour increases, a change in the crumbs’ color also increased, decreasing the degree of saturation and, consequently, the loss in color purity. The highest color saturation value was found in the control bread’s crust, and the lowest in the breadcrumbs 37.5 and 50%.Analyzing the results of color saturation for the bread crust, the control bread obtained the highest average value indicating a more intense color. The bread crumb from the control also obtained the highest average for color saturation, differing statistically  and 32.5% (37.5% and 50% konjac flour bread). Moore et al.  using koThe konjac bread samples had lower protein levels than the bread studied by other authors ,49. WangThe protein content found in 12.5% konjac bread (4.9%) was higher than the other konjac gluten-free bread samples  and sligBread samples with 37.5% and 50% konjac had the lowest levels of lipids. When evaluating the lipid content on formulations based on rice flour, Saueressig, Kaminski, and Escobar  observedAll konjac bread samples obtained lower average carbohydrates than studies of bread with and without gluten by Cornicelli et al. ; Jamiesop < 0.05) was obtained for the moisture content comparing all samples. The highest content was for 50% konjac bread, making up for more than half of the baked bread’s weight, which justifies the lower energy value. Aguiar [A significant difference  obtained a variation between 1.52 to 3.97 cm3/g of bread prepared with corn flour with added fibers. Zelada et al. [3/g in bread samples of corn and rice flour. However, the bread samples with higher SV were not precisely those that presented lower firmness values, as stated by Moore et al. [The bread samples of Moore et al. , using 1d Arendt  obtainedi et al.  obtaineda et al.  obtainede et al.  and Sande et al. , in whicDjordjevic et al.  report tIn formulations of gluten-free bread prepared with rice flour, Nakamura et al.  used konTexture can be defined as the mechanical, geometric, and surface attributes of a perceptible product using instruments and sensory means . The tasAccording to Foste et al. , the strAccording to Turkut et al. , bread cWhen analyzing the results of color saturation for the bread crust, the control bread obtained the highest average value, indicating a more intense color. The control bread crumb also obtained the highest average color saturation. This indicates that, as konjac flour was added to the formulations, the change in the crumb color also increased, decreasing the degree of saturation and, consequently, the loss in color purity. The highest color saturation value was found in the crust of the control bread, and the lowest color saturation in the crumb of the bread 37.5 and 50%.The results for bread crust tonality in this study corroborate the results found in the bread studied by Messa et al. , which aThe weight loss during the baking of the different formulations was lower in the control bread. The moisture content varied between 23.9% and 51.54%. The ash content on konjac bread samples is similar to those found in gluten-free bread from other studies. However, the ash content has increased according to konjac flour addition. It shows that the flour has contributed to the increase of minerals in the composition of the bread. The bread samples with konjac flour showed low caloric values and high fiber contents due to the konjac flour composition.Additionally, they had lower carbohydrate levels, which can positively influence these samples’ glycemic index, but more studies are necessary to evaluate it. Considering the color analysis, the most intense color was obtained in the control bread. As the konjac flour was added to the formulations, the purity of the color was reduced. Konjac flour can be a promising alternative in preparing gluten-free bread because it provided dough mold, growth, and better texture when used in gluten-free bread. The best formulations were prepared in concentrations of up to 37.5% konjac. The 50% konjac bread showed low values for macronutrients, but it was observed that its specific volume was slightly reduced. A limitation of our study is the lack of sensory analysis of the developed bread samples, and further studies are necessary to evaluate their acceptability by consumers.


SEM, and DSC along with co‐culture technique in order to enhance its applicability by evaluating the variations in rheological, textural, morphological, thermal, and structural properties. Our findings suggested that bacterial incorporation both individually and in combination (co‐culture) revealed an improved corn dough profile with better properties. SEM showed irregular shape of particles having more grooves, indentations, and cracks. RVA demonstrated different pasting behavior on the dough. Bacterial inoculation in flour attributed to increase the TO (68.61–71.18), TP (73.74–78.42), TC (78.78–85.36), melting temperature (10.17–15.19), and ΔH (2.72–5.40). The hardness of corn was found approximately 75% of native dough. In treated corn, an increase was noted in both loss and storage modulus in correspondence with changes in the starch configuration and leaching of constituents. The results from DSC presented an increased melting temperature range and gelatinization enthalpy owing to bacterial treatment accredited to diversified morphological characteristics. The outcomes concluded in demonstration of a novel influence on structural, thermal, morphological, and rheological capabilities and capacities of corn dough. Lactic acid bacteria hydrolyzed part of the corn and flour had smaller, irregularly shaped particles with more holes in them, resulting in a reduced water retaining capacity. Textural, thermal, and pasting profile has also been improved due to degradation of macromolecules. Furthermore, the insight alterations induce various changes leading to improved corn flour. It may also develop the associations about the upright insurgence in the corn dough profile and its potential usage in industry and homes.This study is aimed at assessing the effect of lactic acid bacteria (LAB) on corn flour using dynamic characterization methods including RVA, TPA, Rheometer,  L. casei, L. plantarum and L. fermentum on corn flour using dynamic characterization methods including RVA, TPA, Rheometer, SEM, DSC along with co‐culture technique in order to enhance its applicability by evaluating the variations in rheological, textural, morphological, thermal and structural properties.Assessing the effect of  Among them, lactic acid bacteria (LAB) are of particular interest owing to its widespread applications especially in food industry such as yogurt and cheese. It can also be applied as emulsifier, improving agents, and esters due to its hygroscopic nature  is transformed into fodder and alcohol. Owing to certain limitations like low shear and thermal resistance, mucin deficiency, higher retrogradation and thermal decomposition, which limits its functional properties in industry. However, several modifications such as esterification, acidic hydrolysis, carbonation, etherification, polymerization, extrusion, addition of gums, and enzymolysis have been used to improve the applicability of corn flour  were used to improve corn flour properties. The objective of the present investigation was to characterize rheological, morphological, structural, textural, and thermal changes in corn flour due to the inoculation of multiple bacteria (coculture) and single bacteria. The obtained outcomes of the study may provide innovative insights about the corn granules and its deterioration, thereby providing a comprehensive data to improve the quality of a final product.In this study, LAB (2Lactobacillus casei (L. casei), Lactobacillus plantarum (L. plantarum) and Lactobacillus fermentum (L. fermentum) were propagated on MRS medium at 37°C and stored at 4°C. Active dry yeast (Angel brand), sugar and salt were purchased from local market. All other chemicals were of analytical grade.For this study, corn grains (Jingke 968) were obtained from Jilin Agricultural University, China. Milling of the grains was carried out using laboratory miller  the flour was sieved and the fraction retained (0.180 mm) was used. 2.1L. casei, L. plantarum, and L. fermentum were grown in MRS medium (liquid) in an incubator (HZQ‐F160) to a bacterial concentration of 107–108 cfu/mL. Twenty percent bacterial suspension was added in corn flour and placed in incubator for 1–5 days at 37°C. Dough was then prepared by mixing all ingredients thoroughly Table Isolated tor HZQ‐F60 to a b2.2The pasting properties were ascertained by using Rapid Visco Analyzer (Perten) as per the procedure described by Wani  of each sample were obtained at magnifications of ×5000. Flour samples were fixed on the holders with double spread and gold layer was sputtered on it and then scanned in a vacuum of 5kv potential difference.Scanning electron micrographs (Phenom2.4Fifty grams (50 g) of corn flour was kneaded with 50 mL of water in order to make a standard dough by giving 60 min of fermentation at room temperature. The textural analysis comprises hardness, adhesiveness, springiness, cohesiveness, chewiness, gumminess, and resilience were measured by a textural analyzer (TA.XT plus). Maximum force applied can be considered as hardness. The conditions were as follows: test distance 10 mm, probe P/0.5, and test velocity were 0.5 mm.2.5C. The sample was placed on the plate and excessive material was wiped off through the spatula. Silicone oil is added to the sample to avoid the evaporation and 10 min rest was given to equilibrate the stresses. First of all, the linear viscoelastic region was defined through a strain sweep test. Storage (G'), loss modulus (G"), and tangent delta (tan) were determined at constant shear strain and frequency range 0.1–10 Hz.Rheological properties were measured by using rheometer . Frequency sweep test was performed by adopting the following parameters: parallel plates (50 mm), a gap (1 mm), temperature 25°2.6‐1. An empty aluminum pan was used as a reference. The respective parameters were determined with the use of universal analysis 2000 software.Thermal properties of flour samples were analyzed by differential scanning calorimetry . Analysis conditions were as follows: 3 mg sample, 6 μL of distilled water, temperature 30–90°C at a constant rate of 10°C min2.7The obtained data for each parameter were subjected to appropriate statistical analysis through Statistical Package Origin‐Pro 8.5 software. The analysis was performed using completely randomized design (CRD) and the analysis of variance was applied to determine the level of significance.33.1A remarkable variation in textural analysis between simple corn flour and bacterial treated corn flour were observed in this study Table . Bacteri3.2O, peak‐TP, conclusion‐TC, melting temperature TC ‐TO, and gelatinization enthalpy ΔH of flour samples as determined by DSC. Native corn flour showed onset (TO), peak (TP), conclusion (TC), melting temperature (TC ‐TO), and gelatinization enthalpy ΔH were 68.61°C, 73.74°C, 78.78°C, 10.17°C, and 2.72 J/g, which is in line with the reported values by Yangcheng , TP (73.74–78.42), TC (78.78–85.36), melting temperature (10.17–15.19), and ΔH (2.72–5.40). The increasing transition temperatures and gelatinization enthalpy reflected more ordered granules and higher degree of crystallinity, which ultimately results in higher temperatures and leading to delayed gelatinization, which is in agreement with previous studies by Tao who concluded that the higher gelatinization temperature results in delayed gelatinization; ultimately, the granules become more resistant to be gelatinized (Tao et al., Results depicted in Table 3.3The governing factor for proper dough manufacturing is its rheology. A frequency sweep test was conducted to study the quality and processability of dough ingredients. Comprehensive understanding of dough rheology is imperative for the production of a high quality end product (Letang et al., 3.4Pasting parameters of native and bacterial fermented samples were summarized in Table 3.5SEM micrographs permit a direct observation of corn flour to explain the different properties of native and bacterial treated corn flour (Figure r Figure . Bacterir Figure . Smallerr Figure . Bacterir Figure  reported4The study concluded that bacterial treatments, individually and as a coculture modified the quality of flour and has potential applicability. These modifications positively impacted the structural, morphological, rheological, and thermal properties of corn dough. The dough morphology showed more grooves, irregular cracks, and shallow indentations that resulted in a better and more compact dough having good rheological properties. Lactic acid has potential industrial applications and hydrolyzed the flour leading to improved corn dough manufacturing. The results provided deeper insight and sufficient information regarding improvement in corn flour applicability due to bacterial incorporation and it may enhance the acquaintance about the upright revolution in the profile of corn dough and its potential usage in industry and homes.The authors declare no conflict of interest.The manuscript does not involve any human or animal studies and it is according to the guidelines.


Protein tyrosine phosphatases (PTPs) are modulators of cellular functions such as differentiation, metabolism, migration, and survival. PTPs antagonize tyrosine kinases by removing phosphate moieties from molecular signaling residues, thus inhibiting signal transduction. Two PTPs, SHP-1 and SHP-2  and another inhibitory phosphatase, SH2 domain-containing inositol phosphatase (SHIP), are essential for cell function, which is reflected in the defective phenotype of mutant mice. Interestingly, SHP-1, SHP-2, and SHIP mutations are identified in many cases of human leukemia. However, the impact of these phosphatases and their mutations regarding the onset and progression of leukemia is controversial. The ambiguity of the role of these phosphatases imposes challenges on the development of targeting therapies for leukemia. This fundamental problem, confronted by the expanding investigational field of leukemia, will be addressed in this review, which will include a discussion of the molecular mechanisms of SHP-1, SHP-2, and SHIP in normal hematopoiesis and their role in leukemia. Clinical development of leukemic therapies achieved by targeting these phosphatases will be addressed as well. PTPN6 gene, is expressed mainly in hemopoietic cellular systems. SHP-1, with a molecular weight of 68 kDa, is made up of three domains: the N-terminal Src homology-2 (SH2) domain, the C-terminal SH2 domain, and the C-terminal catalytic Protein tyrosine phosphatase (PTP) domain (SH2 domain-containing phosphatase 1 (SHP-1), encoded by the ) domain . Two tyr) domain . The N-S) domain . The “mo) domain . The SHP) domain . In hema) domain .PTPN11 gene, with a molecular weight of 68 kDa, is another member of the PTP family. SHP-2 shares a structure similar to SHP-1: both are composed of two SH2 domains at the N-terminus followed by a PTP domain and two tyrosine residues for phosphorylation by various stimuli at the C-terminus , encoded by the terminus . Comparaterminus . Activatterminus . This reutgrowth . Furtheryndromes  (Shen etyndromes .INPP5 (inositol polyphosphate-5-phosphatase) gene. SHIP interferes with the PI3K (phosphatidylinositol 3-kinase)/Akt pathway by dephosphorylating the P13K product PIP3 to PIP2  is a 145 kDa sized protein that is encoded by the IP2 . In the IP2 . As showIP2 , or it iIP2 . The depIP2 . In addiIP2 . Furtherasophils . Severe asophils . Crohn’sasophils  as well asophils .In this review, we will interpret SHP-1, SHP-2, and SHIP in terms of their association with cell surface receptors, downstream signaling pathways, and roles in hematopoiesis. We will also focus on how SHP-1, SHP-2, and SHIP are involved in hematopoietic malignant diseases, particularly in AML, and the current development of leukemic therapy involving manipulation of their expression.SH2 domain-containing phosphatase 1, SHP-2, and SHIP are recruited by and bound to multiple receptors on the cell surface through their SH2 domains. In myeloid cells, SHP-1 is associated with growth factor receptors, such as c-Kit , and several immunoreceptor tyrosine-based inhibitory motif (ITIM) containing receptors, including paired immunoglobulin-like receptor B (PIR-B), leukocyte immunoglobulin-like receptor 1 (LIR-1), and leukocyte immunoglobulin-like receptor 2 LIR-2; . In hemaSH2 domain-containing phosphatase 1 negatively regulates hematopoietic cell proliferation through many intracellular signals. SHP-1 inhibits cytokine receptors, including Epo-R, IL3-R, and IL-2R, as well as growth factor receptors with intrinsic tyrosine kinase activity, such as CSF-1 and GM-CSF that reduce the proliferation of macrophages and granulocytes . SHP-1 aSeveral receptor tyrosine kinases (RTK) have been identified in hematopoietic cells and are critical mediators of cell signaling . RTKs rein vitro study reported that HSCs from SHP-1 knockout mice have attenuated quiescence and impaired long-term self-renewal  aplasia and lethality in vivo  in the absence of SHIP was significantly lower than wild-type CRUs following transplantation , a mutation present in approximately 30% of AML patients, were compromised by deletion of SHP-1  aimed to enhance the cytotoxicity of the CD33 antibody against AML blasts through epigenetic modifications of PTPN6 , a SHP inhibitor with a potent inhibitory effect on SHP-1, underwent several clinical trials and was proven to treat Leishmaniasis . On the rly 2002 . This stof PTPN6 . Althougin vitro assay of chronic lymphocytic leukemia (CLL), SHP-1 underwent differential phosphorylation and, resultantly, exhibited differential functions and cellular localizations  and myelodysplastic syndrome  translocation, ol group . PTPN11 nomegaly . Given tnomegaly  and SHP0nomegaly  inhibite P2, has been shown to have higher affinity for Akt and led to more potent activation of the Akt pathway in vitro . Treatmein vivo for clinical trials. Furthermore, SHP-2 inhibitors, which have already undergone clinical trials to treat other tumors and cancers, need to be evaluated for their efficacy in the treatment of leukemia. Finally, miR-155 inhibitors, which upregulate SHIP-1, exhibit suppressive effects in AML in vivo and should also be refined for AML clinical trials.The identification of SHP-1, SHP-2, and SHIP and their downstream signaling in hematopoietic cells provides new clues to the development and treatment of leukemia. The studies reviewed here demonstrate that an abnormal level of these phosphatases, instead of altered signaling pathways, is associated with aberrant proliferation of hematopoietic cells and leukemia development. Abnormal expression levels derive either from genetic mutations, such as a loss-of-function mutation of SHP-1 or a gain-of-function SHP-2 mutation, or dysregulated regulatory signals, such as upregulated miR-155 levels that inhibit transcription of the SHIP-1 gene in AML patients. Therefore, SHP-1, SHP-2, and SHIP can be potential targets for anti-leukemia therapy. However, scientists are still not in uniform agreement on the true role of these phosphatases in leukemia, which imposes challenges on the development of leukemia-treating reagents that manipulate expression levels of these proteins. Until now, most studies have supported the oncogenic property of SHP-2 and the tumor suppressor property of SHIP, but the effects of SHP-1 on leukemia are still controversial. Consequently, future studies must focus on confirming the effects of SHP-1 and its downstream pathways in different types of leukemia. In regard to SHP-2, it is necessary to refine its antagonists to suppress leukemia development FH and CW wrote the manuscript and prepared figures. CS provided critical comments and proofreading the manuscript. MC provided comments and joined the discussion. XK organized the structure of the manuscript, provided critical comments and proofreading the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Some of ultrafine-grained (UFG) metals including UFG twinning induced plasticity (TWIP) steels have been found to overcome the paradox of strength and ductility in metals benefiting from their unique deformation modes. Here, this study provides insights into the atomistic process of deformation twin nucleation at Σ3{111} twin boundaries, the dominant type of grain boundary in this UFG high manganese TWIP steel. In response to the applied tensile stresses, grain boundary sliding takes place which changes the structure of coherent Σ3{111} twin boundary from atomistically smooth to partly defective. High resolution transmission electron microscopy demonstrates that the formation of disconnection on Σ3{111} twin boundaries is associated with the motion of Shockley partial dislocations on the boundaries. The twin boundary disconnections act as preferential nucleation sites for deformation twin that is a characteristic difference from the coarse-grained counterpart, and is likely correlated with the lethargy of grain interior dislocation activities, frequently seen in UFG metals. The deformation twin nucleation behavior will be discussed based on in-situ TEM deformation experiments and nanoscale strain distribution analyses results. Similar to many other high strength metals and alloys, UFG metals and alloys commonly exhibit a high strength/hardness that is generally accompanied by poor ductility, known as the strength-ductility paradox, regardless of their crystal structure, for example, UFG aluminum (face-centered cubic)6, UFG iron and interstitial free steels (body-centered cubic)7, and nanograined copper alloys (face-centered cubic)2. The yield strength of materials increases monotonously with decreasing in the grain size, which is generally described by the Hall–Petch relationship9. The tensile ductility drops immediately when the average grain size becomes smaller than 1 µm, which is related to the characteristics of UFG microstructure, that is, fine grains leave very little space for dislocation dynamics leading to a less enhanced strain-hardening rate and resulting in the plastic instability during deformation11.Ultrafine-grained (UFG) and nanocrystalline metals and alloys have attracted intensive research interest since their birth because of their potential to achieve extraordinary properties over conventional coarse-grained (CG) counterparts. This has led to a series of studies regarding the synthesis, processing, characterization, and potential applications of UFG and nanocrystalline metals and alloys in the past few decades12, a UFG Fe–Ni–C metastable austenitic steel13, and UFG high manganese Twinning Induced Plasticity (TWIP) steels16. Experimental results of these materials indicate that nonconventional or reverse-order deformation modes were activated in addition to the normal dislocation slip, specifically, <c + a> dislocations activation in the UFG Mg alloy, martensitic transformation in the UFG metastable austenitic steel, and deformation twinning in the UFG TWIP steels. Tsuji et al.13 suggest that the sequential activation of different deformation modes would foster the regeneration of strain-hardening ability during plastic deformation and lead to a high strength and large ductility because of possible interactions between different deformation modes, in other words, overcoming the paradox of strength and ductility. However, the detailed mechanisms to activate such nonconventional deformation modes are not fully understood yet.Recently, a high strength and moderate ductility are simultaneously achieved in a UFG magnesium alloy14. Our results indicate that the activation takes place in the very early stage of plastic deformation like near the macroscopic yield point, and the grain size plays a major role in deformation mode alternation; deformation twin nucleation occurred at the grain boundaries in under-1 µm austenitic grains, while the normal in-grain slip was mostly observed in over-1 µm grains. This grain size dependence could be attributed to the lack of initial mobile dislocations and inactive in-grain dislocation sources; both are characteristics of the UFG microstructure. Because of its technical and scientific importance, the deformation twinning behavior in conjunction with mechanical properties has been extensively studied in conventional CG TWIP steels19. However, theses deformation twinning mechanisms based on the arrangement of highly coordinated Shockley partial dislocations on {111} slip planes24 appear to be insufficient to explain the grain boundary mediated deformation twinning in the UFG TWIP steels because the deformation twinning in UFG TWIP steels appears to be highly correlated with grain boundary structure.The authors have recently investigated the activation factors of different deformation modes in a UFG austenitic TWIP steel26. Our recent in-situ TEM deformation study has demonstrated that the deformation twin nucleation at a Σ3{111} twin boundary in the CG microstructure occurs only when a localized stress concentration field formed by dislocation pile-up at the grain boundary26. This deformation twin nucleation behavior is unlikely feasible in the UFG microstructure because the grain interior dislocation activities are no longer predominant thus limiting in-grain dislocation pile-up at a twin boundary 27. This leads an open question, that is, what mechanism would promote the deformation twin nucleation at a Σ3{111} twin boundary, which is the dominant boundary type in the UFG TWIP steels, when the localized stress concentration field cannot be generated by dislocation pile-up, or localized stress concentration field is no longer necessary for the deformation twin nucleation in the UFG TWIP steels.Regarding the correlation between grain boundary character and deformation twin mechanism, only handful studies have carried out even on the conventional CG TWIP steels28. The detailed deformed microstructure near Σ3{111} twin boundaries and their local strain level were investigated using transmission electron microscopy (TEM) and scanning transmission microscopy (STEM) techniques to shed a light on the atomistic process of deformation twin nucleation in the UFG microstructure.In this study, we aim to address the question by carefully observing the internal behavior of Σ3{111} twin boundaries subjected to external tensile stresses and revealing the deformation twin nucleation process on the boundaries in a UFG high manganese TWIP steel. A carbon-free UFG high-manganese steel was selected for this study to minimize possible additional effects such as the serrated flow that frequently takes place in carbon-containing TWIP steels−2)29, while the average grain size is about 20 times smaller than that of the CG one, 0.79 µm instead of 15.4 µm. Three grain boundary maps for the undeformed and two different tensile-strained samples are shown in Fig. The microstructure of both undeformed and tensile-strained UFG TWIP steel samples were firstly characterized by EBSD. The UFG TWIP steel has the identical chemical composition and stacking fault energy (SFE) with the CG TWIP steel we previously studied  maps for these three samples are shown in Fig. 31, and in fact, both boundaries were found to act as a nucleation site for deformation twin. The dark contrast indicated by the white arrows appears to be induced by a dislocation reaction within the grain boundary, i.e., a deformation twinning event associated with a grain-boundary dislocation dissociation. In Fig. In Fig. 32. The leading Shockley partial dislocations were continuously emitted from the Σ3{111} twin boundary  turned from dark to bright, i.e., two stacking faults (F1 and F2) were overlapping and showed the reversal fringe contrast. These spontaneously successive emission events occur rapidly at the Σ3{111} twin boundary and is found to be characteristically different from the one induced by in-grain dislocations− Σ3{111} twin boundary interaction in the CG counterpart in Supplementary Fig. S126. A Thompson tetrahedron inserted in Fig. 1 and b2 will glide on the twin plane of Figure fcc zone axis in the inset of Fig. matrix tensile axis causes the resolved shear stress applied on {111} 〈112 〉 leading Shockley partial dislocation to be larger than those applied on {111} 〈110 〉 perfect dislocations and {111} 〈112 〉 trailing Shockley partial dislocations in both Σ3{111} twin plane and deformation twin plane. For example, the Schmid factor for a/6 Σ21 tilt boundary45 or a [011] Σ9 tilt boundary , as shown in Fig. ies Fig. c–e, was 50 was applied as schematically illustrated in Fig. t1 to t8, i.e., the vectors from t1 to t4 and the vectors from t5 to t8 were defined with respect to the crystal frame of grain I (Twin) and grain II (Matrix), respectively. The Burgers vector of twin boundary dislocation can be determined by the summation of the lattice vectors when lattice vectors of t5 ~ t8 were coordinately transformed into the ones in crystal frame of twin. The Burgers vector of the twin boundary dislocation then can be expressed as the following:1 ~ t8 are listed in Fig. 1 and d3 are the DSC lattice vectors illustrated in the schematic of Fig. To determine the Burgers vector of the twin boundary dislocations associated with the twin boundary disconnection, the Burgers circuit approach52. In contrast, the disappearance of localized strain field in Fig. A quantitative analysis of nanoscale strain distribution indicates the correlation between local strain level and deformation twinning behavior. The strain level at/near the disconnection in Fig. 54. As a result, these disconnection propagations on Σ3{111} twin boundary results multiple disconnections, i.e., the atomistic step having few monolayers in height, on the originally atomistically smooth twin boundaries, that accommodates the misfit between two sides of grains across the twin boundary. The multiple disconnections emitted sequentially from triple junction and coexisted on the neighboring {111} twin plane are similar to the internal behavior observed by in situ atomistic resolution TEM experiment54 where a Σ11(113) coherent grain boundary showed a layer-by-layer migration behavior due to the sequential nucleation and lateral motion of grain boundary disconnections. This disconnection activity combination with shear coupled grain boundary migration was also observed in a nanocrystalline gold metal by in situ TEM deformation experiments and considered to be the dominant deformation mechanism55. The disconnections can act as preferential nucleation sites for stacking faults because the localized stress concentration field at/near disconnections could exceed the twinning stress during plastic deformation, thus allow in-grain dislocation slip transfer in the later stage of plastic deformation56. This grain boundary sliding process appears to be phenomenologically similar to the grain boundary migration mechanism originally proposed by Hirth57 and experimentally observed in the deformed microstructure of several nanograined coppers59.The mechanism to form a nucleation site for deformation twins is schematically illustrated in Fig. bR is the Burgers vector of residual twin boundary dislocation, bin is the Burgers vector of the dislocations within a disconnection, bout is the Shockley partial dislocation emitted from the disconnection. Based on the crystallographic orientation relationship between the Σ3{111} twin boundary and deformation twin, bin and bout can be designated to be 1/6 Σ9), and low-energy coherent Σ3{111} twin boundaries are all able to be a nucleation site for deformation twins.The formation of twin boundary disconnections appears to be governed by the motion of Shockley partial dislocations on the Σ3{111} twin boundary. These disconnections (defective steps) would accumulate strain and serve as the nucleation sites of deformation twins when the localized stress level exceeds the twinning stress.The mechanistic difference to generate localized strain concentrations on coherent Σ3{111} twin boundaries between the UFG and the CG alloys is concluded to be originated from a unique characteristic of the UFG microstructure. The grain size constraint suppresses the in-grain dislocation activities in the UFG steel and stimulates the twin boundary dislocation activities leading to the twin boundary disconnections which act as the deformation twin nucleation sites. On the other hand, the observed periodic contrast reversal of stacking fault during the deformation twin nucleation at Σ3{111} boundaries indicates that the deformation twin formation process is likely identical between the UFG and CG microstructures once nucleated, partly due to the identical chemistry and SFE.The deformation twin nucleation mechanism on Σ3{111} twin boundaries in a UFG Fe-31Mn-3Si-3Al austenitic TWIP steel was found to be characteristically different from that in the CG counterpart. The detailed microstructural investigation using in-situ TEM deformation, HRTEM and µP-STEM techniques has drawn the following conclusions:−2) was fabricated for this work29. The chemical composition of the steel was Fe-31Mn-3Al-3Si wt%. The as-received TWIP steel was cold rolled from 12 to 1 mm (92% reduction) by multi-pass cold rolling and then heat treated in a salt bath at 950 °C for 5 min followed by water quenching to obtain the aimed grain size.A carbon-free UFG (0.79 ± 0.39 μm) high-manganese austenitic steel TWIP steel with intermediate SFE  and transmission electron microscopy (TEM) techniques. The 150 µm thick sheets were tensile-deformed to 0.046 and 0.062 engineering strain using a testing machine (Kammrath and Weiss Module 200 N) at a strain rate of 4.6 × 10Specimens for post-deformation EBSD analyses were mechanically polished by abrasive paper up to 2000-grit then electropolished. The microstructural features including grain boundary maps and local strain distribution were examined using a TSL OIM EBSD system attached to a FEI Helios 600 dual-beam field emission gun (FEG) scanning electron microscope (SEM) / focused ion beam (FIB) system. EBSD maps were acquired at 30 kV acceleration voltage and 13 mm working distance. The scanning step size was 50 nm. No data clean-up was performed except for the removal of some points with low confidence value.The local strain distribution was evaluated from the kernel average misorientation (KAM). KAM is a local misorientation defined as the average misorientation of a point with its nearest neighbors in a grain. The average misorientation of a given point is calculated by taking that point as well as all of its nearest neighbors into account with a criterion that the misorientation exceeding threshold 3° will be discarded from the calculation, because these points are considered to belong to the adjacent grains.Specimens prepared for post-deformation TEM analyses were cut from the center of the deformed sheets. 2 × 2 mm square shape foils were taken then mechanically thinned to 70 μm thick. The final thinning to be partly electron transparent was achieved by using a twin-jet electropolisher (Fischione Model 110) with a 95% acetic acid—5% perchloric acid electrolyte maintained at 17 °C and the applied voltage of 38 V. The TEM characterization was performed using a JEOL JEM 2100 operated at 200 kV with Gatan Orius SC200D and Ultrascan 1000XP cameras.60 after thinned to be partly electron transparent. The strain rate was controlled at approximately 6.7 × 10–5 s−1 in this study. All videos were recorded in the bright field (BF) mode and a FEI Titan 80–300 S/TEM operated at 300 kV was used. Gatan Orius SC200D camera and Digital Micrograph with the high-resolution streaming video plug-in were used for video recording.Specimens for in-situ deformation tests were sliced from undeformed sheets; the shape of the foils, 2 × 2 mm square shape, is identical to the one used for TEM analysis. A square shape foil was fixed on a cartridge-type blade for SATO Holder Duo (Mel-Build Co.)The FEI Titan 80-300 V S/TEM also was used in this analysis. Strain mapping was performed using the parallel-nanoprobe scanning transmission electron microscopy (µP-STEM) technique. The µP-STEM employed a 10 µm second condenser aperture to reduce the beam semi-convergence angle to be 0.13 mrad and obtained less than 2 nm probe size.Supplementary Information 1.Supplementary Video 1.


Electrical-assisted (EA) forming technology is a promising technology to improve the formability of hard-deformable materials, such as Mg alloys. Herein, EA micro tensile tests and various microstructure characterizations were conducted to study the electroplastic effect (EPE) and size effect on the mechanical responses, deformation mechanisms, and fracture characteristics of AZ31 Mg foils. With the assistance of electric currents, the ductility of the foils was significantly improved, the size effects caused by grain size and sample thickness were weakened, and the sigmoidal shape of the flow stress curves during the early deformation stage became less obvious. The EBSD characterization results showed that the shape change of the flow stress curves was due to the EPE suppressing the activation of extension twinning at the early deformation stage, especially for the coarse grain samples. The suppression of extension twinning resulted in a quick increase in flow stress due to the dislocation-dominant work hardening, and the increased flow stress eventually promoted extensive deformation twins at large deformation. Thus, as the sample strained to 10% tensile deformation, the EA-tested samples showed a larger volume fraction of deformation twins than the non-EA samples. The reference orientation deviation analysis verified that the deformation twins in the EA samples were formed in the large deformation stage. Combined with the fractography, the EPE also improved the ductility by suppressing the expansion of cleavage surfaces. To coordinate the deformation in the c-axis, c-axis of parent grains around 86.3° [Magnesium (Mg) alloys are of growing interest in micro-parts for medical purposes and microelectromechanical systems (MEMS) for their high specific strength and excellent biodegradability . Howevernd 86.3° . Moreovend 86.3° ,6,7.Electrical-assisted (EA) plasticity is regarded as an effective way to improve the formability of hard-deformable metals ,10,11,12It is commonly accepted that the mechanisms behind the EPE on the deformation and mechanical properties of metals mainly includes the Joule heating effect and the athermal current-induced effect. Troitskik et al.  firstly The plastic deformation and formability of Mg alloys are rather sensitive to the intrinsic microstructure feature and external boundary constraints . For insd) of the samples annealed at 450 °C for 10 h was about 21 μm (coarse grain) and the grain size of the samples annealed at 250 °C for 2 h was about 9 μm (fine grain). The annealed samples were cut into miniature plate dog bone-shaped specimens with thicknesses t = 0.5 mm and 1.0 mm, as shown in The as-received material was a commercial extruded AZ31 Mg alloy. It was refined by equal channel angular pressing (ECAP) operations and annealed under different heat treatments to obtain samples with various grain sizes. The grain size  pulse currents of varying intensities, i.e., J = 10, 30, 40, and 50 A/mmHowever, the size effects caused by grain size and sample thickness were weakened with the assistance of electric currents. The sigmoidal shape feature of the flow stress curves during the early deformation stage was attenuated with the increase in current intensity. Therefore, it can be inferred that the electric current suppressed deformation twinning at the early deformation stage. Since the activation and expansion of extension twinning can release the stress concentration during the plastic deformation, once the extension twinning is suppressed, the work hardening resulting from the dislocation slip will be enhanced. Furthermore, one can see that the yield strength increased with the assistance of electric currents, especially for the coarse grain samples in a,b; the t = 0.5 mm . Grain boundary (GB), as a crystal defect, exhibits a higher electrical resistance than the interior grains during the EA deformation. The volume fraction of GB increases with the decrease in grain size, and more localized Joule heating will be generated at the GBs during the EA deformation [t = 0.5 mm, the elongation was increased by approximately 60% when the assisted electric current density was 50 A/mm2.In contrast, the ultimate tensile strength (UTS) is rather sensitive to the current intensity. Though there is a common rule that the UTS decreases with the increase in current intensity in this study, the EPE on the UTS of the samples with different thicknesses and grain sizes was quite different. For the coarse grain samples with = 0.5 mm a, when t= 1.0 mm b, all thormation . Thus, a2) samples with different grain sizes and thicknesses. The black lines represent high-angle GBs  and the band contrast (BC) maps with GBs and twinning boundaries (TBs) are shown in Comparing the EA coarse grain sample b with thBased on the EBSD data shown in To quantitatively describe the influence of size effect and the EPE on the deformation heterogeneity of the Mg foils, the KAM histogram of the deformed samples is shown in To verify the possible roles of the EPE in affecting dislocation slip and deformation twinning, the grain reference orientation deviation  maps of the deformed samples were computed from the EBSD data, and the results are shown in To reveal the EPE on the deformation modes, we further compared the maps of the crystal directions of the misorientation axes of the non-EA and EA coarse grain samples with thickness e region . The whiThe fracture characteristics of the non-EA and EA uniaxial tension with different sample thicknesses and different grain sizes were examined and compared, and The electroplastic effect (EPE) significantly influenced the mechanical responses of AZ31 Mg foils with different grain sizes and sample thicknesses during the EA micro-tension tests. The size effects caused by grain size and sample size were weakened with the assistance of the EPE. The coarse grain samples are more sensitive to electric currents during the tension tests as the yield strength increased, although the ultimate tensile strength decreased. The EBSD characterizations demonstrate that only extension twins were activated during the non-EA and EA tension deformation and the electric current suppressed the activation of extension twins at the early deformation stage, especially for the coarse grain samples. However, as the deformation increased, the flow stress due to the dislocation-dominant work hardening was increased, which eventually resulted in the activation of extensive deformation twins at large deformation. Thus, the EA-tested samples showed a larger volume fraction of deformation twins than the non-EA samples after they were strained to 10% tensile deformation. The reference orientation deviation analysis verified that the deformation twins in the EA samples were formed in the large deformation stage. Meanwhile, the EPE can promote damage tolerance and prevent the expansion of the cleavage surfaces; thus, the formability of the EA tension samples was increased during the EA tension tests. To improve the reliability of EA forming technology in the manufacturing of micro-scaled products, advanced constitutive models considering the size effect and the EPE must be established based on current research, especially the combined effect on coordinating various deformation mechanisms such as dislocation slip and deformation twinning.


Intravascular processing of triglyceride-rich lipoproteins (TRLs) is crucial for delivery of dietary lipids fueling energy metabolism in heart and skeletal muscle and for storage in white adipose tissue. During the last decade, mechanisms underlying focal lipolytic processing of TRLs along the luminal surface of capillaries have been clarified by fresh insights into the functions of lipoprotein lipase (LPL); LPL’s dedicated transporter protein, glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1); and its endogenous inhibitors, angiopoietin-like (ANGPTL) proteins 3, 4, and 8. Key discoveries in LPL biology include solving the crystal structure of LPL, showing LPL is catalytically active as a monomer rather than as a homodimer, and that the borderline stability of LPL’s hydrolase domain is crucial for the regulation of LPL activity. Another key discovery was understanding how ANGPTL4 regulates LPL activity. The binding of ANGPTL4 to LPL sequences adjacent to the catalytic cavity triggers cooperative and sequential unfolding of LPL’s hydrolase domain resulting in irreversible collapse of the catalytic cavity and loss of LPL activity. Recent studies have highlighted the importance of the ANGPTL3–ANGPTL8 complex for endocrine regulation of LPL activity in oxidative organs , but the molecular mechanisms have not been fully defined. New insights have also been gained into LPL–GPIHBP1 interactions and how GPIHBP1 moves LPL to its site of action in the capillary lumen. GPIHBP1 is an atypical member of the LU (Ly6/uPAR) domain protein superfamily, containing an intrinsically disordered and highly acidic N-terminal extension and a disulfide bond–rich three-fingered LU domain. Both the disordered acidic domain and the folded LU domain are crucial for the stability and transport of LPL, and for modulating its susceptibility to ANGPTL4-mediated unfolding. This review focuses on recent advances in the biology and biochemistry of crucial proteins for intravascular lipolysis. LPL or its partners  severely impair the efficiency of triglyceride hydrolysis, causing lifelong severe hypertriglyceridemia—the familial chylomicronemia syndrome  called chylomicrons . Anothersyndrome . Chylomisyndrome . Heterozsyndrome . In contsyndrome . For exasyndrome . Similarof ASCVD .In the past decade, the prevailing model for LPL-mediated TRL processing has been transformed by new discoveries on the function and dynamics of proteins that participate in LPL transport (GPIHBP1) and help control LPL activity (ANGPTLs). In this review, we focus on the dynamic interplay between LPL, GPIHBP1, and ANGPTLs 3, 4, and 8. We will emphasize the importance of transient interactions between these proteins and discuss the relevance of protein disorder and marginal protein stability to the regulation of LPL and to the compartmentalization of its activity in the intravascular unit. We define the intravascular unit as (i) the capillary endothelial cell, (ii) the subendothelial spaces containing extracellular matrix molecules, and (iii) the parenchymal cells (myocytes and adipocytes) adjacent to capillaries .Discovered in 1943 as a heparin-releasable clearing factor , lipoproThe appearance of LPL in evolution predates teleosts; it is found in all vertebrates and is highly conserved in mammals, with 58–99% sequence identity at the amino acid level . LPL is sn-1/sn-3 ester bonds of triglycerides in the neutral lipid core of chylomicrons and VLDL, thereby releasing two unesterified fatty acids and a sn-2 monoacylglycerol . Of note, the tryptophan-rich loop of one LPL protomer occludes the catalytic cleft of the partner protomer, raising doubts about whether the homodimer configuration in the crystal structure is enzymatically active  with an oxyanion hole (Trp55 and Leu133) and a lid region (residues 217–238) constrained by a Cys216–Cys239 disulfide bond. LPL was in an open lid configuration, presumably because the catalytic cleft was occluded by the Trp-rich loop of the PLAT domain (residues 387–394)  required for correct folding in vivo and for restoring LPL activity during refolding in vitro (174) had been uncovered in a kindred with chylomicronemia  spanning the N- and C-terminal domains and extending across the interdomain interface  . Hydroge387–394) . The cryin vitro . Interescronemia . This miom cells . The crynterface . This stnterface .Years ago, sedimentation equilibrium analyses of purified preparations of bovine LPL strongly suggested that LPL was a homodimer . Subsequin vitro as judged by the rapid exchange of LPL protomers under conditions that favor interactions between LPL molecules  of 34.8°C—below normal body temperature , the calnexin and calreticulin cycle probably also plays a role in LPL quality control . The functional relevance of dual tethering by SDC-1 and sphingolipid-rich secretory vesicles was supported by experiments with an LPL mutant harboring a dysfunctional Trp-rich loop. Secretion of that LPL mutant was inefficient but was restored when the mutant was co-expressed with GPIHBP1 , a Ca2+-coordinating amino acid , the lid region , and LPL’s binding interface with GPIHBP1 . The crystal structure of LPL has been useful in understanding how LPL mutations affect function  helps stabilize LPL and preserve its structure and activity. Several genetic variants associated with hypertriglyceridemia are located in the interface between the N- and C-terminal domains of LPL .Genetic studies have uncovered more than 100 eridemia . The prefunction . As noteLPL polymorphic variant  results in a truncated LPL protein lacking the last two residues (Ser-Gly). Interestingly, this variant has a gain-of-function phenotype characterized by reduced plasma triglyceride levels, increased pre- and post-heparin LPL levels, and reduced risk of cardiovascular disease  domain ; this extension undergoes posttranslational O-sulfation of Tyr18  domain . GPIHBP1ur exons  rather tof Tyr18  and is de anchor .58   removal of N- and C-terminal signal peptides responsible for secretion and glycolipid anchoring, respectively, (ii) N-linked glycosylation of Asn58 , (iii) Oof Tyr18 , and (iv–Cys116) . The fol–Cys116) . It bindnterface . This binterface  and sitenterface —and it enterface  as they BCL11A into GPIHBP1  analyses, (vi) its fast hydrogen-deuterium exchange profile, and (v) its absence from the crystal structure of the GPIHBP1•LPL complex  and moderate dissociation rates (koff of 1 × 10–2 s–1)  studies is far from trivial because LPL is unstable and prone to aggregation. Nonetheless, available SPR studies agree that GPIHBP1 has two distinct interactions with LPL—a fast and transient electrostatic interaction between the acidic domain and LPL’s heparin binding sites and a slow and stable interaction between the folded LU domain and LPL’s PLAT domain . In one 0–2 s–1) . That mo0–2 s–1) . The ult on koff . This ob on koff .in vitro  is increased dramatically by GPIHBP11–131 binding (Tm of 57.6°C) but only marginally by GPIHBP134–131 binding (Tm of 37.7°C), underscoring the pivotal role of the acidic domain in this process ; (ii) the C-terminus of LPL, where truncation of two residues (p.Ser447X) is associated with beneficial effects in population studies; and (iii) the PCSK3 (furin) cleavage site in LPL (residues 296–299). The susceptibility of LPL to PCSK3 cleavage is impacted reciprocally by ANGPTL4 and GPIHBP1 binding . In tissues from –/–Gpihbp1 mice, LPL is neither uniformly distributed throughout the sub-endothelial spaces nor swept away by the lymph drainage. Under these conditions, LPL is surprisingly more abundant around the abluminal surface of capillaries (–/–Col18) have moderate hypertriglyceridemia and reduced plasma levels of LPL  (GPIHBP1 variants with missense mutations affecting cysteine residues give rise to multimerized GPIHBP1 molecules on the cell surface that do not bind LPL (107Cys) with a free unpaired thiol group  . In cellbind LPL , 2015. Iol group . Mice thl spaces . Despite89) to interact with LPL  α and PPARγ . ANGPTL451 and Cys55  connected to a C-terminal fibrinogen-like domain by a linker region . The thrnd Cys55 . These sin vitro . A diffe trimers . This st trimers . Neverth trimers . This obbona fide proprotein convertase cleavage motif  of 0.9–1.7 μM (Ki of 3.2 nM), but the reaction kinetics still followed a reversible non-competitive inhibition model , while the presence of ANGPTL4 lowers Tm by 20°C to less than 15°C for LPL alone and to 36.6°C for LPL–GPIHBP1 complexes (t1/2 ∼180 s) and is metastable in the presence of ANGPTL4 (t1/2 < 5 s), while LPL in LPL–GPIHBP1 complexes is stable and is borderline stable in the presence of ANGPTL4 (t1/2 ∼70 s). This dependency would favor preservation of GPIHBP1-bound LPL activity along the capillary lumen.Lipoprotein lipase inhibition by ANGPTL4 is abrogated at 20°C when LPL is bound to GPIHBP1, but this effect is less pronounced at 37°C . The temomplexes . At norm1–33 and GPIHBP134–131 do not effectively preserve LPL activity, either alone or in combination  and a C-terminal fibrinogen-like domain connected by a linker region  or by PCSK6 (on the cell surface) at its proprotein convertase cleavage motif located in the linker region  resembles an ANGPTL4 variant (p.Asp15Lys or E40K) inasmuch as both SNPs eliminate the negative side-chain in the start of the α-helix harboring the LPL binding motif  that interferes with ANGPTL3s ability to inhibit LPL activity  and anotTL3 mRNA . So far,TL3 mRNA .The last decade has brought an increased clarity into the molecular mechanisms driving intravascular lipolysis. This new insight combines three-dimensional protein structures and their conformational dynamics with the compartmental regulation of LPL activity in the vascular unit across tissues. Despite this advancement, the next decade will likely bring additional new insights into the mechanism by which the ANGPTL3-ANGPTL8 complex regulates LPL activity and how ApoA5 modulates this regulation.MP wrote the manuscript with inputs from all authors. KK prepared the figures. All the authors contributed to the article and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.