Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length  Cells are complex systems that rely on numerous tightly regulated vital processes. For instance, protein quality control is crucial for maintaining the cell’s proteome. To avoid the lethal accumulation of misfolded or nonfunctional proteins, eukaryotes as well as prokaryotes use proteolysis . In thisAAA+ proteases belong to the family of ATPases associated with various cellular activities and are molecular machines capable of unfolding and degrading proteins . AAA+ prEscherichia coli, FtsH is the only one that is anchored to the membrane and that is essential   unfolded proteins can reach the translocating loops and be moved through the pore for degradation  part , 19, 20 cryo-EM) . Therefocryo-EM) , two- (1cryo-EM) , 15, or cryo-EM)  symmetricryo-EM) . In contima FtsH , 15, andcus FtsH , 18. Theradation .A. aeolicus FtsH (AaFtsH), which we determined with negative stain electron microscopy to a resolution of 20 Å and with cryo-electron microscopy to resolutions of 6.6 Å with sixfold symmetry and 16 Å without the symmetry imposed. Unexpectedly, upon detergent solubilization we found not only AaFtsH hexamers but also fully stable and active AaFtsH dodecamers. The dodecamer structure from negatively stained specimen was solved to a resolution of 25 Å, showing two AaFtsH hexamers sharing a single lauryl maltose neopentyl glycol (LMNG) micelle. This arrangement induces a tilt of the periplasmic domain with respect to the cytosolic domain. In this conformation, the periplasmic domain of one hexamer touches the cytosolic domain of the other hexamer. Cryo-EM analysis revealed a variety of S-shaped or V-shaped dodecameric structures with differing N-terminal interactions at resolutions from 12.3 to 20.5 Å.Here we report the first full-length structure of Since both AaFtsH hexamers and dodecamers have similar ATPase and proteolytic activities, we propose that the cytosolic domain tilts with respect to the membrane plane so that substrates can reach the translocating pore loops, as required for substrate unfolding and degradation. Such a large conformational change relates to the unique properties of the 20-aa linker between the end of the second transmembrane helix and the ATPase domain. Eliminating this linker leads to inactive AaFtsH hexamers that are not able to form dodecamers.E. coli cells and extracted from purified membranes with the use of a mild detergent, LMNG  and a second peak at 13.4 ± 0.2 ml  (A). The peak positions were determined by simultaneously fitting two Gaussian functions. Native gel electrophoresis shows that the second peak has an approximate molecular weight of ∼700 kDa, whereas the first peak indicates a larger complex (B). The molecular weight of the eluted complexes was estimated using the partition coefficient (Kav) values extracted from a calibration curve of the Superose 6 column and the positions of fitted Gaussian functions. The second peak is centered at a molecular weight of 730 kDa, which is larger than the size expected for AaFtsH hexamers (∼430 kDa), as expected owing to the weight contribution of the bound detergent micelle. On extrapolation of the calibration curve to smaller elution volumes, the first peak corresponds to a molecular weight of 940 kDa.AaFtsH with a C-terminal His-tag was overexpressed in nt, LMNG . Ni-NTA nt, LMNG . Best re N = 10) A. The pe complex B. The moTo determine more precisely the molecular weight of AaFtsH oligomers and LMNG micelles, size-exclusion chromatography combined with static light scattering (SEC-MALS) was used. SEC-MALS accounts for the amount of detergent bound to a membrane protein and allows for a determination of the molecular weight of a protein in a protein-detergent mixed micelle . SEC-MALC).Transmission electron microscopy (TEM) was used to visualize samples from each SEC peak. Negative stain EM analysis of particles from the second peak, with molecular weight compatible with AaFtsH hexamers, exhibits structures with an average length of 154 ± 13 Å  C.A–D displays representative side or tilted views of AaFtsH hexamers. Using 10 class averages from AaFtsH hexamers, we measured that the estimated length for the full hexamer is 167 ± 5 Å  and its width is 131 ± 7 Å . When compared with the dimensions of A. aeolicus FtsH cytosolic crystal structure, a similar width is reported (120 Å) (E), has a thickness of 40 ± 4 Å, which is close to the lipid bilayer thickness, and width of 100 ± 18 Å.2D Class averages of the negative stain preparation were calculated from 15,000 images of AaFtsH hexamer particles using the image processing software packages Scipion1.1  and EMAN (120 Å) . The cyt (120 Å) . Finally (120 Å) E, has a F displays the 20-Å resolution 3D map of the AaFtsH hexamer, which accommodates the crystal structure of the AaFtsH cytosolic domain (PDB 4WW0) and the E. coli FtsH periplasmic domain . Albeit the resolution achieved is only 20 Å, the orientation of the cytosolic domain with respect to the detergent micelle is clearly visible. Some of the hexamer classes show that the cytosolic domain is tilted in relation to the detergent micelle, creating a gap for substrate entry (A–D).From the 2D class averages, EMAN2.12 calculated starting models and refined one of them against all particles, imposing a sixfold symmetry. Figure 2E) using Relion 3.0.8 showed a large structural variability. In full-hexameric classes, the well-formed cytosolic domain displays high-resolution features , whereas the N termini are fuzzy (H). Owing to a small number of particles remaining in the dataset after 3D classification (5649), 3D refinement was initially performed in C6 symmetry. The acquired 3D map revealed a protein structure similar to the negatively stained one, with clearly discernible cytosolic and N-terminal (periplasmic + transmembrane) domains . From this ring, six symmetric densities extend further upward. Each of them then makes an outward U-turn of 2 × 90° and subsequently joins the cytosolic domain again on the outer surface. The crystallized 4V0B structure of the periplasmic domain could be fitted in the top of the U-turn density in the expected position between the two transmembrane helices , suggested that the symmetry constraint should be released.In order to investigate the dynamics of the structure, we performed a local resolution analysis in Relion, which indicated that the resolution varied from the highest 6.6 Å in the periplasmic domain to less than 10 Å in the cytosolic domain. This result, together with the presence of the closed channel in the cytosolic chamber I, suggesK). It possesses a distorted cytosolic domain, which could still be fitted with six individual 4WW0 subunits. Its N-terminal domain is fully disordered, and only one subunit seems to partially follow the folding of the C6-symmetric structure. All six N termini bundle into an indistinguishable, tilted off-axis mass, which was already observed in the negatively stained class averages (A–D). The N termini do not form any inner ring; instead, they give rise to a wide opening between the membrane and the cytosolic domain (K) for substrate entry.Therefore, we also resolved the structure of the AaFtsH hexamer without imposing symmetry at 15.9 Å resolution, using only 2129 particles K. It posaverages , A–D. Thc domain K for subA. aeolicus and the YME1 protease are highly similar, we attempted to fit the 6AZ0 model of the YME1 cytosolic domain into the cytosolic domain of FtsH cryo-EM map. As a result, the FtsH map and the YME1 model shared the asymmetric appearance induced by the nucleotide-dependent conformational changes .Finally, we attempted to find out if the cytosolic domain of the asymmetric FtsH cryo-EM map possesses the staircase arrangement observed in other AAA ATPases , 27 withD). Particles from the first peak appear to house two AaFtsH hexamers, hence corresponding to a dodecamer, although their average length is less than twice the average length of the hexamer particles.Negative stain EM was also performed on the first SEC peak revealing elongated particles with an average length of 231 ± 15 Å . To obtain a 3D map of the dodecamers, class averages that correspond to a straight (B–D) rather than a V-shaped conformation (A) were selected to calculate an initial model that was subsequently refined against all projections of these classes without imposing any symmetry. F–G illustrate the resulting 3D map and indicate how the structure of the AaFtsH cytosolic domain (PDB 4WW0) fits. All class averages and 3D maps suggest that the AaFtsH dodecamer is kept in solution by a single LMNG micelle that embraces 2 × 12 transmembrane helices (green in E), whereas the periplasmic domains of each hexamer contact the cytosolic domains of the other hexamer .2D Class averages were calculated using 15,000 images of AaFtsH dodecamer particles yielding class averages showing the conformational variability of AaFtsH , A–D. Tostraight , B–D ratormation A were segreen in E, whereaA–D). A gap as large as 20 Å is observed between the central pore of the AaFtsH cytosolic domain and the detergent micelle, and a gap of ∼30 Å between the edge of that domain and the micelle . From the initial dataset of 101,726 particle images identified by Gautomatch picking (F), 41,818 remained after several Relion 2D classification runs. They were submitted to a 3D classification, which identified 25% of hexameric, 72% of dodecameric particles, and 3% misshaped particles. From the dodecameric particles, 36% contributed to two dodecameric structures connected at angles close to 90°, and 64% to one of three classes of S-shaped dodecamers (I), which seem to be composed of two opposing AaFtsH hexamers with tilted N-terminal domains.The dodecamer structures resolved with cryo-EM confirm the structural variability observed in negatively stained 2D class averages H. From t picking F, 41,818decamers I, which I) further accentuated the structural variability by assigning their particles into many low-resolution classes unsuitable for midresolution refinement. The final dataset of the S-shaped classes, comprising 16,749 particle images, could be split into three major classes with final counts of 4351, 2651, and 1722 particle images, which could be refined to resolutions of 17, 19.5, and 20.5 Å, respectively . These three classes strongly differ in the structure of the micellar domain, which can be described as “intertwined” (17 Å), “lamellar” (19.5 Å), and “touching” (20.5 Å). Owing to the low resolution, it is impossible to trace the course of individual N-terminal domains in the micellar part. On the other hand, one of the cytosolic domains is always better structured than its counterpart, which suggests further structural variability within the class averages. The length of the S-shaped AaFtsH dodecamers is about 290 Å.Subsequent 3D classification of both V-shaped dodecameric classes  were fitted using initial velocities calculated at various ATP concentrations (A–B), with the use of FtsH monomer concentrations in the calculations  and a Kcat = 63 min−1 (95% CI: 58–69 min−1). The dodecamer fraction has a KM = 518 μM (95% CI: 432–625 μM) and a Kcat = 61 min−1 (95% CI: 57–66 min−1), identical to the values for the hexameric AaFtsH within error limits. ATPase activity was measured at 60 °C. The optimal growth temperature of A. aeolicus is 85 °C (ATP hydrolysis rates were assessed by measuring the inorganic phosphate (Pi) released. The initial velocity of the ATP hydrolysis reactions is extracted from the concentration of Pi released in the first 10-min interval . From thtrations , A–B, wiulations , 31. The95% CI: 4–625 μM ais 85 °C .C) and the dodecamer  fractions. The hexamers and the dodecamers reaction rates both increased linearly with AaFtsH concentrations at a slope of ∼1.8 nM of resorufin per minute per micromole of AaFtsH. This validates the idea that both fractions have the same proteolytical activity, suggesting that their active centers are not only well folded but also equally accessible for protein entrance.Next, we assessed the proteolytic activity of the hexamer and the dodecamer forms of AaFtsH. As previously reported , 33, we Fig. 4To prove that the oligomeric states of AaFtsH are fixed over the course of the experiment, the SEC fractions from the hexamer peak and the dodecamer peak were collected and incubated with protease buffer for 30 min at 60 °C, mimicking the protease activity assay conditions. After the 30-min incubation, both fractions were reanalyzed by SEC. Our SEC result indicates that AaFtsH hexamers and dodecamers remain in their oligomeric states, at least over the course of 30 min (duration of protease activity assay), as shown in the E. coli and A. aeolicus FtsH protein sequences and mapped the available crystal structures onto the sequence alignment (A). Basic Local Alignment Search Tool (BLAST)   finds th (BLAST)  shows 13 (BLAST) .To assess the importance of the flexible linker between the TM and the AAA domain, we constructed the AaFtsH-Δ20 mutant, in which a genomic sequence corresponding to a 20-aa linker (residues 123–142) was deleted. SEC-MALS experiments show that AaFtsH-Δ20 eluted in a single peak with a molecular weight of ∼416 kDa corresponding to a hexamer ; this assembly was further confirmed by negative stain EM . In contA. aeolicus FtsH and characterized its structure with negative stain and cryo-EM. The purification of full-length FtsH hexamers proved to be challenging, with size exclusion chromatograms reproducibly showing two species of AaFtsH in closely overlapping fractions. SEC-MALS and electron microscopy image analysis confirmed that the low-molecular-weight fraction corresponds to AaFtsH hexamers and the high-molecular-weight fraction to AaFtsH dodecamers  and Kcat . The KM values obtained for ATPase activity of hexamers and dodecamers are between 7× (E. coli FtsH solubilized in a different detergent and under different conditions. Both fractions of LMNG-solubilized AaFtsH can degrade a casein substrate at comparable proteolytic rates (C–D). Our results also document that AaFtsH hexamer and dodecamer both show high proteolytic activity, since the same amount of casein can be degraded by 10x less LMNG-AaFtsH when compared with AaFtsH solubilized in n-dodecyl-β-D-maltoside  rendered at 20-Å resolution fits well with the cytosolic domain of the 3D map presented here. There is a similar match of the periplasmic domain with the periplasmic crystal structure of E. coli FtsH (PDB 4V0B). The reported intermembrane domain of m-AAA is larger  are also structurally highly variable, in particular considering the different arrangements of the N-terminal domains. Together with the well-resolved periplasmic domain (I), this indicates an amazing flexibility of the connector between periplasmic and cytosolic domains, providing FtsH with the capability to process a wide variety of substrates.The unexpected AaFtsH dodecamers , which exhibits a length of ∼13 Å in its quiescent state but can extend like a spring at least up to ∼30 Å, as the structure predictors show . The space between the cytosolic domain and the tilted membrane compares with the height of the AaFtsH periplasmic domain. When FtsH adopts the tilted conformation, enabled by the flexible linker, both soluble and membrane-bound substrates would reach the translocation pore more efficiently (C–D).Based on these results we propose a new model for substrate entry. Conformational variations demonstrated by both AaFtsH hexamers and dodecamers  and 3 iniciently , C–D.in vivo. A higher-resolution model of full-length FtsH would give us more details on how FtsH interacts with its substrates. This has, however, proven to be difficult, precisely owing to the high flexibility observed in the different particles present on EM grids.Although it is unlikely that FtsH forms head-to-tail dodecamers in the bacterial membrane, we propose that the large tilting movements observed here are likely achieved during proteolytic activity A. aeolicus FtsH (AaFtsH) cloned into pET22a vector was kindly provided by Ulrich Baumann and the same expression conditions as previously reported were employed  LMNG (Anatrace) for 3 h at 4 °C and cleared at 125,000g for 1 h at 4 °C. The sample was first purified by affinity chromatography, using a HisTrap-5 ml column . FtsH fractions were eluted in 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.01% (w/v) LMNG, and 200 mM imidazole. AaFtsH fractions were then incubated at 60 °C overnight with 20 mM ATP, 10 mM MgCl2, and 25 μM ZnCl2. The incubated sample was concentrated to 500 μl and loaded into a SEC Superose 6 Increase 10/300 GL column  pre-equilibrated with 10 mM Tris-HCl pH8.0, 150 mM NaCl, 0.01% (w/v) LMNG, and 5% glycerol. AaFtsH monomer concentration was measured with a Nanodrop. A representative SEC run is plotted in Figure 1A. The center position of the two largest peaks was determined by fitting Gaussian functions to 10 SEC profiles. A calibration curve of the Superose 6 Increase 10/300 GL was performed using the Gel Filtration High Molecular Weight Calibration Kit , following the GE Healthcare instructions. AaFtsH fractions were analyzed by Native PAGE gels using the MiniPROTEANTGX Precast Protein Gels (Bio-Rad).Full-length employed . Cells w2, 5 mM MgCl2, 1 mM dithiothreitol, 0.01% LMNG, and 10 mM ATP) at 60 °C for 30 min. After that incubation period both fractions were reloaded into a SEC Superose 6 Increase 10/300 GL column  separately, the SEC column was pre-equilibrated with 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% (w/v) LMNG, and 5% glycerol. The resulting SEC profile is in To check the oligomeric state of AaFtsH over the course of the activity assays, the fractions corresponding to the first (dodecamers) and second (hexamers) fractions were incubated with the protease activity assay buffer . A dn/dc value of 0.14 ml/g was used for LMNG , 42. FtsThree microliters of AaFtsH hexamer or dodecamer fractions were loaded on a carbon-coated 400 square mesh copper grid (Aurion) previously glow discharged for 1 min. The liquid drop was absorbed with filter paper after 1 min and quickly washed with a drop of water that was again blotted with filter paper. This procedure was repeated three times to rinse all the detergent present in the samples. Finally, a 3-μl drop of 3% uranyl-acetate was added to the grid, incubated for 1 min and absorbed with a filter paper. TEM was performed using a Philips CM-200T and a JEOL 3200 FSC both equipped with a TemCam-F416 (TVIPS) and recorded at 50,000× magnification using the EM-MENU software with a sampling rate of 2.23 Å/pixel.A. aeolicus cytosolic domain (PDB 4WW0), the cryo-EM of the full-length m-AAA (EMDB 1712), and the crystal structure of the E. coli periplasmic domain (PDB 4V0B) were automatically fitted into our 3D map. To this end, the high-resolution structures were rendered to the resolution of the respective 3D maps. The fit of PDB 4WW0 to the FtsH cytosolic domain yielded a correlation of 0.6. Fitting the periplasmic crystal structure of E. coli (PDB 4VOB) to the periplasmic region achieved a correlation of 0.9. Similar correlation values were obtained when fitting two PDB 4WW0 to the 3D map of the FtsH dodecamer. The higher correlation values for the fits for the 3D maps of negatively stained samples compared with those for the 3D maps of vitrified samples relate the lower resolution of the former maps.Single particle imaging processing was performed using Scipion1.1  and EMAN−/Å−2 for hexamers and dodecamers, respectively. AaFtsH hexamers were imaged at a magnification of 47,259× (1.058 Å/pixel) and saved as 50-frame super-resolution stacks, dodecamers were imaged at a magnification of 78,247× (0.64 Å/pixel) and saved as counted stacks of 35 frames.Three microliters of the purified AaFtsH samples at a concentration of 0.7 mg/ml were applied to a Quantifoil R1.2/1.3 (hexamers) or Quantifoil R2/2 300 mesh grid (dodecamers), glow discharged for 30 s prior to freezing. The grids were plunge frozen in liquid ethane with a FEI Vitrobot Mark IV (Thermo Fisher Scientific) under these conditions: temperature, 4 °C; humidity, 95%; blotting time, 2 s; and blotting force set to 1. Frozen grids were imaged in a Titan Krios TEM  equipped with a Gatan Quantum-LS energy filter (20-eV zero-loss filtering) and a Gatan K2 Summit detector. Images were acquired with SerialEM program  at totalThe acquired movies were processed during the imaging session with the Focus program , which ihttps://www2.mrc-lmb.cam.ac.uk/research/locally-developed-software/zhang-software/#gauto). All calculations were performed by Relion 3.0.8. Selection of particles from aligned micrographs was done in two steps. Initially, particles from 100 randomly selected images were semiautomatically selected with EMAN2  and subjA total of 35,048 AaFtsH particles were extracted from the aligned micrographs; 7635 particles were selected for subsequent 3D classification and refinement. In sixfold-symmetric processing, 5649 contributing to well-folded 3D classes were subjected to final 3D refinement, yielding a structure with 6.6-Å nominal resolution. Without an imposed symmetry, 3D classification revealed only one major class with 2129 particles, which could be refined to 15.9-Å resolution. A spherical blob with half-width of 140 Å was used as the initial 3D model.via their “tilted” N-terminal domains could be refined to resolutions of 17 Å (4351 particles), 19.5 Å (2651 particles), and 20.5 Å (1722 particles).A total of 101,726 particles were extracted from the dodecamer dataset; 41,818 were submitted to 3D processing with the same spherical blob serving as initial model. Five dodecameric structures, one hexameric structure, and one “rubbish” structure were identified in 3D classification with seven classes. Each of the dodecameric classes was further processed, only three “S-shaped” classes where the two AaFtsH hexamers seemed to be connected A. aeolicus, better fit could be achieved when positioning the six 4WW0 subunits individually. The asymmetric 6AZ0 model of the YME1 protease was fitted in full length.All rigid-body fitting was performed in UCSF Chimera. Although the resolved sixfold-symmetric hexamer structure could accommodate two copies of the 4WW0 trimeric FtsH crystal structure of 2), was previously incubated with PiBind resin (Innova Bioscience). Reactions were started by mixing AaFtsH  in mix A with 50, 100, 250, 500, 1000, or 1800 μM ATP . Reaction rates were measured every 2 min for a total of 10 min, in triplicates. ATP concentrations were chosen such that the maximum concentrations were more than 10× the previously reported KM values  microplate reader; every replica was measured three times and an average of these readings was calculated for each replica . Free phosphate from the AaFtsH dodecamers and hexamers was eliminated by incubation with 100 μl of PiBind resin (Innova Bioscience) for 30 min. Also mix A  at 60 °C. Triplicates of these measurements were taken. Reaction rates were measured every 10 min for 30 min  plate reader.AaFtsH protease activity was assessed using the resorufin-labeled casein kit (Roche); 0.25, 0.5, 1.0, or 2.0 μM  of AaFtsH hexamers or dodecamers were incubated with 50 μM of resorufin-labeled casein in   . Residuenih.gov) .Cryo-EM structures of AaFtsH hexamers and dodecamers have been deposited under accession numbers EMD-11161, EMD-11167, EMD-11169, EMD-11171, EMD-11200, and EMD-11201. Other data from this study are available from the corresponding authors upon request.The authors declare that they have no conflicts of interest with the contents of this article.


Growth factor receptor-bound 2 (Grb2) is an important link in the receptor tyrosine kinase signaling cascades. It is involved in crucial processes, both physiological (mainly embryogenesis) and pathological (different types of cancer). Several binding partners of all three domains (SH3–SH2–SH3) of this adaptor protein are well described, such as ErbB family members for the SH2 domain and Sos for the SH3 domains. How the different domains interact with each other, both structurally and functionally, is still unclear. These interactions could be essential for regulation processes, and therefore are of great interest. Although a lot of structural data on Grb2 exist, they describe either individual domains, ligand-bound conformations, or frozen pictures of the protein captured by crystallography. Here we report the assignment of backbone and of  Growth factor receptor-bound 2 (Grb2) is an adaptor protein essential for early steps of embryogenesis, especially differentiation  and of the CSH3 free domain (PDB 1GFC) were solved by NMR soon after Grb2 identification  was inserted into a pETM11 vector also containing the gene conferring resistance to kanamycin. The construct contains a N-terminal His6 tag followed by a TEV protease cleavage site for protein purification. Grb2 plasmid was transformed into BL21 star(DE3) Cell lysis was obtained by disruption (Constant Systems Ltd.), the lysate was ultracentrifuged and the supernatant filtered before loading on a HisTrap FF crude column . After elution, the His6 tag was removed by TEV cleavage, leaving two additional residues G and A at the N-terminus compared to the native Grb2 sequence. The final protein product therefore had 219 residues. Excess of TEV protease, free tag and uncleaved protein was removed with a Ni-NTA resin (Thermo Scientific), and purification was completed with gel filtration on a Superdex-75 column . Despite deuteration during expression and given that the purification was performed in Spectra were recorded at 308K with Bruker Avance NMR spectrometers of proton frequencies 600, 800 and 950 MHz equipped with TCI cryoprobes. Backbone and Overall structural integrity of each domain  was assessed using chemical shifts and TALOS+ CO spectra allowed assignment of 86% of all Additional peaks in the spectra  The isolated NSH3 domain of Grb2 has been shown to exchange between a folded and an unfolded conformation under acidic conditions  has been shown to undergo a similar equilibrium in physiological buffer , a few tens of peaks are present that we excluded as belonging to any unassigned residue. They are mainly located in the central part of the spectrum in the proton dimension, suggesting that they are part of an unfolded conformation. Moreover, some of these peaks could be assigned to small segments consistent with parts of the NSH3 domain sequence (assignment not reported here). Our hypothesis is that these additional peaks correspond to an unfolded form of the NSH3 domain in full-length Grb2, the other two domains being only present in their fully folded form. From peak volumes, the unfolded to folded molar ratio is of the order of 1:1.Data deposition The http://www.bmrb.wisc.edu/) under the BMRB accession number 50082.


UBE3 genes, the biochemistry of UBE3 enzymes, their biology and clinical relevance in brain disorders. Particularly, we highlight that uninterrupted action of UBE3 ligases is a sine qua non for cortical circuit assembly and higher cognitive functions of the neocortex.Protein ubiquitination belongs to the best characterized pathways of protein degradation in the cell; however, our current knowledge on its physiological consequences is just the tip of an iceberg. The divergence of enzymatic executors of ubiquitination led to some 600–700 E3 ubiquitin ligases embedded in the human genome. Notably, mutations in around 13% of these genes are causative of severe neurological diseases. Despite this, molecular and cellular context of ubiquitination remains poorly characterized, especially in the developing brain. In this review article, we summarize recent findings on brain-expressed HECT-type E3 UBE3 ligases and their murine orthologues, comprising Angelman syndrome UBE3A, Kaufman oculocerebrofacial syndrome UBE3B and autism spectrum disorder-associated UBE3C. We summarize evolutionary emergence of three  Primata. Spatiotemporally synchronized patterns of activity in and between neuronal circuits are enabled by tightly controlled transcriptional–translational and morphoregulatory programs in developing neocortex [Uninterrupted communication between executive brain centers, on a molecular and single neuron level manifested as circuit activity, allows for integration of external and internal sensory stimuli and generation of appropriate efferent responses. The neocortex, paramount achievement of mammalian brain evolution, constitutes a biological substrate for higher cognitive abilities in eocortex .Assembly of a neuronal circuits in the mammalian neocortex starts during early neurogenesis, where at the ventricular zone (VZ) of the neural tube, radial glial cells (RGCs), give rise to postmitotic cells that differentiate into glutamatergic excitatory neurons. One of the most prominent features of cortical neuron development is that the immature nerve cells born close to the VZ do not stay in situ but migrate radially to distribute horizontally into their target niches to establish neocortical layers. These cortical niches contain neurons originating from spatiotemporally defined RGC populations. Neurons of each layer share transcriptional signatures and display similar physiological properties and patterns of connectivity. Molecular signaling orchestrating the postmitotic differentiation is conserved between neurons of different layers. The initial transcriptional ground states in RGCs, often display very subtle differences, but represent spatiotemporally restricted developmental trajectories that seem to be the source of the excitatory neuronal subtype variations in the cerebral cortex ,3. FormaAberrant assembly of neuronal circuits represents the primary cause of higher cognitive dysfunction . MalfuncNeuronal transcriptional signatures are the effect of a discrete regulation of gene expression. Recent reports derive the sources of cortical neuron complexity using state-of-the-art single cell transcriptomics to cluster the types of cellular populations present in the developing brain. These elegant but somewhat reductive approaches turn a blind eye to the involvement of post-transcriptional modes of gene expression regulation in the brain development. Recent accumulating evidence reveals critical roles of post-translational modification of ubiquitination in the formation of neuronal networks ,10.Since its discovery in 1975 , ubiquitProtein ubiquitylation is reversible and catalyzed by ubiquitin proteases, enzymes called deubiquitylases (DUBs). Human genome harbors some 90 loci encoding for DUBs, far less than for E3 ligases, indicating a rather broad biochemical promiscuity. However, some DUBs display a strong ubiquitin chain type affinity, i.e., they catalyze proteolytic cleavage of certain polyUb chains, rather than the ubiquitylated substrate per se .Given a distinct mode of catalysis, E3 ligases fall into two, or three main classes. Homologous to E6-AP (HECT) type E3 Ub ligases act as direct Ub acceptors, conjugating it to their C-terminal cysteine residue, before shuttling the Ub onto their substrates. HECT is a group of biochemically distinct enzymes with 28 members in humans identified to date. Our previous works signify the involvement of HECT-type E3 ligases in the most fundamental neurodevelopmental processes including establishment of dendritic arbor , neuriteClassically, ubiquitination of a substrate protein has been linked to its degradation. Loss-of-function mutations in E3 ligases are associated with neurological diseases, implicating the pivotal role of specific degradation in fundamental neurodevelopmental pathways ergo cortical circuit assembly. Recent data sheds lights onto non-degradative ubiquitination, encoded by polyUb chain type conjugated to the substrate, indicative of these types of modifications at play during neuronal development. The K48- and K63-linked polyUb chains constitute the major types of polyUb chains in the mouse brain and human frontal cortex and K11-linked chains are three- to four-fold less abundant . CanonicWhereas monoUb has been linked to modulating enzymatic activity or protein–protein interactions , differeThe determinants of chain type formed by a given E3 Ub ligase have been an area of debate. Regarding the HECT ligases, these are the function of the catalytic HECT domain and do not seem to depend on the E2 conjugating enzyme, but rather are located within the 60 amino acids of the C-terminus of the HECT domain C-lobe . Within Understanding of neuronal roles of UBE3s requires an in-depth analysis of the biochemistry of their action. Transient binding of Ub (autoubiquitination) prior to its sequential transfer onto the substrates, is a distinct feature of HECT-type ligases. Evolutionarily conserved organization of the catalytic C-terminal HECT domain highlights its E2 conjugating enzyme-interacting N-lobe, a short flexible linker and a C-lobe, containing an Ub acceptor cysteine . ConformWe know a fair amount regarding the organization of UBE3A HECT domain and its mode of action . BiochemUBE3B leading to single amino acid substitutions in the HECT domain appear to affect the enzymatic activity of the ligase implicating conformational restrictions. Theoretical modeling of Q727P substitution predicts it renders the HECT domain unfit for substrate binding and positioning towards catalytic cysteine, required for efficient ubiquitination [Ube3b conditional forebrain-specific knockout (cKO) neurons, and the latter one exhibits altered subneuronal localization, when compared to the native UBE3B [KOS-associated point mutations in tination . HECT-love UBE3B . It is pve UBE3B . The sceRecent studies have shed light on the crystal structure of UBE3C . Aside fThere has been a slight confusion about the functional homology between UBE3s, as observed in introductions to multiple publications. All three UBE3s share a gross domain organization plan including the catalytic HECT domain and long stretches of low complexity, possibly accounting for the high interactivity of ligases with binding partners . UBE3B aUbe3 genes. These expression trajectories indicate that each mRNA is present in progenitors and postmitotic neurons in the developing murine neocortex [Single cell RNA sequencing databases provide insights into spatiotemporal expression dynamics of eocortex .Ube3s dates long before the origin of the central nervous system. The history of Ube3a seems more tumultuous than that of Ube3b and Ube3c. Orthologues of Ube3a are identified in bilaterians, including vertebrates and fruit fly, but not in C. elegans indicating Ube3a loss in some nematodes [Ube3a shows secondary loss in choanoflagellate lineage. On top of this, the origin of Ube3a imprinting seems to be acquired in the mammalian radiation. Ube3b appeared relatively early in evolution and is present in fungi and other opisthokonta. Of the three Ube3s, Ube3c seems to be the youngest with its orthologues found in Bilateria and other Metazoa [Ube3s and their orthologues, implicated in human neurodevelopmental disorders, which seem to regulate key cellular housekeeping processes in primitive organisms [Phylogenetic analyses show that HECT E3s appear before animals or very early in their evolution and appearance of them precede the emergence of the cellular function that they control . That saematodes . Additio Metazoa   with, among other symptoms, severe intellectual disability, speech impairment, behavioral disturbances, and developmental delay [UBE3A, is a genetic cause of the ASD [UBE3A in our previous review [Ube3a KO in mice (Ube3am−/p+) recapitulates features of AS patients. Loss of Ube3a leads to ineffective LTP in the hippocampus, indicative of defects in neuronal circuitry associated with learning and memory [The gene encoding for the founding member of HECT type ligase family, E6-associated protein, al delay . Duplica the ASD . We summs review . In humas review . Maternad memory .UBE3A is located on chromosome 15, the mode of AS inheritance is not typical autosomal dominant or recessive [UBE3A and mouse Ube3a are silenced by large non-cording antisense RNA, expressed from the nearby loci [Ube3a-ATS) is transcribed by an atypical RNA polymerase type II and covers the entire Ube3a locus. Deletion of maternal Ube3a causes the complete lack of Ube3a protein expression, restored by additional targeting of paternal Ube3a-ATS [Ube3a-ATS is a critical suppressor of the Ube3a expression from the paternal allele, which is likely the case in humans as well. Interestingly, Ube3a-ATS is expressed solely in neurons, without affecting expression of Ube3a in glial cells [While ecessive ,50. Paterby loci . The murbe3a-ATS . Ube3a-Aal cells ,53. Of nal cells ,55.T485A reduces proteasome activity and leads to a stabilization of β-catenin, indicative of its enhanced ability activate Wnt signaling [UBE3A regarding the assembly of cortical circuits for the sake of its functional comparison with UBE3B and UBE3C.Other reviews  highlighignaling . The T48ignaling . UBE3A hignaling . As is tignaling . Quite iignaling . Upon inignaling . There iUbe3a downregulation leads to less complex dendritic arbors in the cortex and hippocampus and abrogates growth of apical dendrites associated with loss of polarization of Golgi apparatus. Such phenotypes are reversible by re-expression of isoform 2, but not of isoform 1 or 3 of Ube3a [Isoform- and tissue context-specific morphoregulatory roles of UBE3A have been fundamental for our understanding of its contribution to the development and functions of neuronal circuits. Five isoforms of UBE3A mRNAs were identified with three of the transcripts coding for protein isoforms . Ube3a dof Ube3a . In contof Ube3a . Anotherof Ube3a . Moreoveof Ube3a .Expression of different isoforms of UBE3A has its relevance in the pathophysiology of AS. Developmental well-being of AS patients seem to rely on the isoforms of UBE3A affected in the genome. For instance, patients with Met1Thr mutation eliminating isoform 1 of maternal UBE3A exhibit far more advanced developmental skills as compared to other non-mosaic AS patients .Ube3am−/p+ mice exhibit increased PTPA level and protein phosphatase 2A (PP2A) activity. Normalizing PTPA levels, as well as pharmacological inhibition of PP2A reversed developmental defects of excitatory synapses in AS mice [Drosophila [Ube3a localizes at the axon terminals and at the head and neck of dendritic spines  to regul AS mice . Regardi AS mice . BMP reposophila .UBE3A involvement in a developmental syndrome presenting a severe ID, a significant body of literature addresses synaptic roles of the ligase. Recently, extensive local synaptic actions of Small Ub-Like Modifier 1 (SUMO1), have been rebutted since in neurons, SUMO1 was found mainly in the nucleus [Ube3a KO mouse [Ube3a loss-linked sociability deficits [Given  nucleus . Recent KO mouse . Mimickideficits . It is pdeficits .2+- and voltage-dependent big potassium (BK) channels in human neurons and brain organoids. Inhibition of BK channels in AS mice alleviates the seizure susceptibility demonstrating the channelopathy as underlying cause of AS network dysfunction [In line with this, UBE3A suppresses neuronal hyperexcitability by degradative ubiquitination of Cafunction . Moreovefunction .Ube3a, but not its juvenile or adult loss, recapitulates all behavioral deficits of AS mice [Ube3a is responsible for perturbed excitation/inhibition imbalance with enhanced seizure susceptibility, signifying inhibitory neuron-specific loss of Ube3a as a principal reason for hyperactive neuronal circuits in AS mouse model [Neuron-restricted imprinting of UBE3A offers ways of intervention in AS mouse models, which have been useful to determine that pharmacological un-silencing of paternal allele in the adulthood is possible ,55, yet  AS mice . There s AS mice , or exci AS mice . GABAergse model .As mentioned before, maternal copy number gains of 15q11.2–q13.3 lead to Dup15q syndrome, characterized by intellectual disability, developmental delay, muscle hypotonia and speech impairment. Dup15q represents the most penetrant chromosomal anomaly in ASD patients accounting for 1–3% cases worldwide. UBE3A belongs to approximately 40 genes in that chromosomal region and its gain-of-function is thought to represent the molecular cause in Dup15q patients . Indeed,In 1971, Robert Kaufman described four of seven siblings, who exhibited severe mental retardation with absent speech, characteristic facial dysmorphisms with blepharophimosis, aberrances of the eye, mongoloid slant of the palpebral fissures and small mandible. Already then, an autosomal recessive mode of disease inheritance seemed the most likely , later cUBE3B, which inherited in autosomal recessive manner, are causative for KOS [Initially, two consecutive reports have revealed loss-of-function mutations in  for KOS ,93. Sinc for KOS ,94.UBE3B gene was discovered as a gene upregulated after acoustic trauma in the chick basal papilla. Its expression dramatically increased in the lesion in chick inner ear, but not in undamaged regions [UBE3B generates two splice isoforms, one devoid of the HECT domain, presumptively non-functional [ regions . Human Unctional . Notablynctional . Some ofnctional . FurtherOverall, UBE3B is highly expressed in the central nervous system, digestive tract, respiratory system, as well as in multiple cell lineages of skin and other soft tissues. Severe developmental delay in KOS patients and multi-organ defects signify the involvement of UBE3B in systemic homeostasis. Similarly to KOS, G>A polymorphism in cattle resulting in exon skipping and in-frame deletion of a portion of catalytic HECT domain is associated with ptosis, intellectual disability, growth retardation and increased mortality . BesidesUBE3B loss-of-function was discovered causative of KOS [Soon after e of KOS ,93, the e of KOS . Contrare of KOS , variatie of KOS .2+ are especially relevant in the light of UBE3B implications in the stress response and cellular viability. In the first paper identifying mutations in UBE3B as a cause of KOS, researchers demonstrate that C. elegans ortholog of UBE3B, oxi-1 is a player in cellular protection under oxidative stress [The findings on UBE3B-mediated mitochondrial function and intracellular Cae stress . Moreovee stress . Additioe stress . Given aUBE3B as a candidate ASD gene in a whole-exome sequencing and homozygosity analysis of 16 probands. Additionally, Ube3b mRNA level increased in E16.5 mouse cortical neurons at DIV6 (day-in-vitro 6) after KCl-induced hyperpolarization, again linking UBE3B, neuronal excitability and Ca2+ [Ube3b in mice leads to severe developmental defects including anatomical, behavioral, and metabolic disturbances, including growth retardation, hypoplasia of corpus callosum, enlarged ventricles and decreased cortical thickness. Using Golgi–Cox labeling, authors show that cortical neurons in Ube3b−/− mice display reduced dendritic branching and spine density. Using a cell line stably expressing tagged UBE3B, affinity chromatography and mass spectrometry, authors identify branched-chain α-ketoacid dehydrogenase kinase (BCKDK) as a putative UBE3B target, further validated in ubiquitination assays. BCKDK level was also increased in Ube3b−/− cortex, liver and skeletal muscle, indicative of a specific pathway for UBE3B-mediated BCKDK degradation. Consequently, authors demonstrate that metabolite profiles of Ube3b−/− mice display signs of severe metabolic perturbations, with high levels of, among others, plasma spermidine, and S-adenosylhomocysteine, and cortical urea. Authors also show that patients bearing compound heterozygous mutations in UBE3B display similar metabolic perturbations, as identified in mice. Mitochondrial physiology, as demonstrated before in a cell line, is affected in skeletal muscle of Ube3b−/−, but not in the liver. That is particularly interesting, given some indications of UBE3B indispensability for skeletal muscle function [Ube3b−/− mice include loss of vocalizations, decreased grip strength and decreased grooming and nesting. The interpretation of behavioral changes is, however, not straightforward, as severe developmental and anatomical defects, likely account for poor sensorimotor performance of the Ube3b−/− animals and occlude the behavioral readouts of Ube3b-associated neuronal deficit-linked changes. Despite these limitations, full Ube3b KO mice are a model of human systemic KOS, a metabolic disease strongly affecting the skeletal and nervous system [The next report on UBE3B function focuses on its role in the metabolic pathways and links it to the development of the central nervous system. In their previous report, this group of researchers first identified and Ca2+ . Next, tfunction . Behavios system .Ube3b−/− mice, Emx1-driven, forebrain neuron and glia-specific mouse model was established, given a high expression of Ube3b in the cortex and the hippocampus. Ube3b associates with postsynaptic density fractions and regulates dendritic branching in a cell-autonomous manner. Conditional deletion of Ube3b in the forebrain is associated with decreased cortical volume, due to reduction of dendritic trees, and thinning of corpus callosum. Neurons and glia-restricted loss of Ube3b leads to increased spine density, a phenotype reversible by re-introducing Ube3b by in vivo transfection. Increased density of spines is in line with increased frequency of miniature excitatory postsynaptic potentials (mEPSPs). Regarding neuronal physiology, Ube3b maintains the ratio of NMDA to AMPA at the synapse and abundance of NR2A. Forebrain-restricted loss of Ube3b leads to altered activity of hippocampal circuit and induces complex behavioral changes. These include specific hippocampal circuitry-associated loss of spatial memory and increased social memory. Moreover, Ube3b cKO male mice exhibit a behavioral switch towards self-directed actions (like grooming and eating) at the expense of exteroceptive behaviors, such as exploration. On a molecular level, with regard to its synaptogenic function, Ube3b ubiquitinates γ-subunit of calcineurin, Ppp3cc and regulates its levels at the synapse [Our most recent work on the murine ortholog of UBE3B addresses specifically its cell-autonomous role in developing nervous system and involvement in formation of neuronal circuits. To circumvent the syndromic lethality and the overt non-neuronal phenotypes in the  synapse . Among t synapse . Future Ube3b−/− mouse model serves to study human KOS, it is not clear if the brain phenotypes described for the full KO animals are due to a loss of Ube3b or constitute secondary effects of systemic dyshomeostasis and/or other endophenotypes. The forebrain-specific Ube3b cKO does not serve as a comprehensive model of KOS, since it restricts the loss of the gene to excitatory neurons and glia, but offers a possibility to determine the cell autonomous roles of Ube3b in the cerebral cortex and the hippocampus. Notably, of the most significantly increased metabolites in Ube3b−/− was urea, whose elevated levels are associated with brain inflammation, simplified dendritic arbors and reduced spine density in a mouse model of kidney failure [Ube3b−/− mice and address the kidney-brain axis regarding the systemic influence of uremic metabolites on the developing neurons. It is tempting to speculate that loss of spines and synapses in Ube3b−/− mice is due to a non-cell autonomous mechanism. This should be verified in experiments addressing the effect of neuronal exposure to metabolites identified in plasma and cortex of Ube3b−/− mice on spine density. Additionally, future experiments should address the effects of calcineurin and/or other protein phosphatase inhibition on synapse number both in Ube3b conditional and conventional mouse models. This is particularly thought-provoking regarding the UBE3A-driven regulation of PP2A activity and small molecule-mediated rescue of AS-like phenotypes in Ube3am−/p+ mice.The case of Ube3b and the abovementioned two reports reopens a discussion on the applicability of genetic mouse models and conclusions drawn based on neurodevelopmental phenotypes. While the  failure . Further2+-binding messenger, inhibits the enzymatic activity of Ube3b. On the other hand, Ube3b ubiquitinates and/ergo regulates the level of the γ-subunit of calcineurin, Ca2+-, and calmodulin-dependent serine/threonine protein phosphatase. Taken together, neuronal depolarization and Ca2+ are important regulators of Ube3b enzymatic activity in the developing neocortex, and in turn, the enzymatic activity of Ube3b controls the cellular responders to Ca2+ , or KIAA10. Since its identification through cDNA sequencing in myeloid cells , relativpatients ,40 (Tablpatients . Future UBE3C with disorders such as asthma [The involvement of UBE3C in various types of cancer has been most commonly reported. In many cases, upregulated UBE3C promotes the progression of the disease by tumorigenic and metastatic effects, associated with an increase in cellular migration and proliferation ,121,122.s asthma , stroke s asthma , major ds asthma ,126, cocs asthma , Parkinss asthma , and Dows asthma .UBE3C seems to have a distinct biological role as an E3 ligase that directly influences the cellular proteolytic machinery. Firstly, UBE3C and a DUB Usp14 continuously cycle on and off 26S proteasomes in ubiquitinated substrates-dependent manner and regulate cellular proteostasis . UBE3C hAside from its well-established role in proteolysis involving its inherent ability to conjugate K48-linked polyUb , UBE3C fDespite classical grouping into one family, UBE3 ligases display fundamentally different characteristics, supported by the phylogenetic, biochemical, physiological, and clinical evidence. Each of UBE3 constitutes a separate biochemical entity, with specific ubiquitination pathways that they supervise, necessary for neuronal development. Based on the recent evidence, UBE3A has been shown to operate in the nucleus in an isoform-specific manner, where it globally regulates neuronal homeostasis. Other isoforms of UBE3A seem to have opposing effects in terms of their morphoregulatory and synaptogenic potential, and might operate locally in neurons, some of them not necessarily as enzymes. On top of this, UBE3A seems to modulate cellular proteolytic machinery. UBE3B associates with synaptic fractions, possibly forming a bigger protein complex, involving calcineurin. It would be interesting to test if its metabolic role is achieved in a global manner, by operating in different cellular compartments, similarly to UBE3A. Proteasome function depends on the association with ASD-linked UBE3C, able to assemble non-canonical polyUb chains. It would be extremely important to address developmental roles of not solely the ligase, but also of proteasome, its activity and non-degradative ubiquitination in ASD mouse models.Future research will bring answers not only in terms of detailed molecular context of their action, but also regarding the biochemistry of non-conventional ubiquitination and its cellular and physiological consequences. This will aid clinical trial design for neurological and neurodegenerative diseases and cancer and shed light onto the complex machinery at play in the development and homeostasis of neuronal circuits.


Caenorhabditis genomes harbor two uggt gene copies that exhibit differences in their properties.UDP- glucose: glycoprotein glucosyltransferase (UGGT) is a protein that operates as the gatekeeper for the endoplasmic reticulum (ER) quality control mechanism of glycoprotein folding. It is known that vertebrates and uggt genes went through independent duplications in Caenorhabditis and vertebrates. In both lineages, the catalytic domain of the duplicated genes was subjected to a strong purifying selective pressure, while the recognition domain was subjected to episodic positive diversifying selection. Selective relaxation in the recognition domain was more pronounced in Caenorhabditis uggt-b than in vertebrates uggt-2. Structural bioinformatics analysis revealed that Caenorhabditis UGGT-b protein lacks essential sequences proposed to be involved in the recognition of unfolded proteins. When we assayed glucosyltrasferase activity of a chimeric protein composed by Caenorhabditis uggt-b recognition domain fused to S. pombe catalytic domain expressed in yeast, no activity was detected.Bayesian phylogenetic inference based on 195 UGGT and UGGT-like protein sequences of an ample spectrum of eukaryotic species showed that Caenorhabditis, uggt-b evolved under different constraints compared to uggt-a which, by means of a putative neofunctionalization process, resulted in a non-redundant paralog. The non-canonical function of uggt-b in the worm lineage highlights the need to take precautions before generalizing gene functions in model organisms.The present results support the conservation of the UGGT activity in the catalytic domain and a putative divergent function of the recognition domain for the UGGT2 protein in vertebrates, which would have gone through a specialization process. In  Approximately one-third of all cellular proteins are imported into the lumen of the ER or integrated into its membranes and most of them are glycoproteins. The ER retains some of these proteins while others are exported into the secretory pathway . The ER N-glycosylation of proteins starts with the addition of a triglucosylated glycan to nascent polypeptide chains by the oligosaccharyltransferase complex. Concomitantly, this triglucosylated glycan is processed by glucosidase I and glucosidase II (GII) (The II (GII) . MonogluII (GII) . This enII (GII)  Cycles oUGGTs are monomeric soluble proteins composed of at least two domains . The N-tHUGT1 and HUGT2)  worms only show subtle deleterious phenotypes as those found in mutant worms that lack both CNX and/or CRT  .We analyzed a total of 195 UGGT (and UGGT-like) protein sequences retrieved from Genbank (https://parasite.wormbase.org). To get this final set of sequences we initially retrieved the list of all nematodes orthologs of uggt-a and uggt-b from Wormbase. Using the Wormbase stable IDs we retrieved orthologous coding sequences with BioMart. Duplicate sequences and those that did not start with a Methionine were excluded from the final set. The sequences of two highly related rhabditid genera, Oscheius and Diploscapter were included to test the monophyly of Caenorhabditis UGGTs. As occurred with Caenorhabditis sequences, we retrieved almost identical sequences in Diploscapter coronatus which belonged to different scaffolds that do not represent a duplication event, but rather represent intra-individual (haplo-genomic) variation of a diploid genome .A total of 47 UGGT nematode sequences were retrieved using BioMart via the Wormbase ParaSite database  but were excluded from the phylogeny and selection analysis since we decided to compare duplicated copies.Within uggt-a and uggt-b resulted reciprocally monophyletic but together did not form a monophyletic group, we ran nucleotide and protein phylogenies to test their relationships within nematodes, confirming uggt-a and uggt-b are paraphyletic as described in Results section.Phylogenetic analyses were run in MrBayes 3.2.6 (7 Markov Chain Monte Carlo (MCMC) generations, sampling every 1000 generations. A suitable burnin fraction was selected based on the resulting Estimated Sample Size (ESS) which was below 0.01 by 21.65% of the MCMC chain, where it showed as well a stationary trace distribution. The UGGT sequence of the grass species Oryza brachyantha was set as outgroup.We then ran the definitive analysis fixing the Jones model for 1.3x10Caenorhabditis. Gap rich positions were removed with GBlocks 0.91b  to synonymous substitutions (dS). To this end, we performed protein alignments for each separate UGGT of vertebrates and Caenorhabditis. Separate alignments were performed for each protein domain. Regions spanning UGGT recognition and catalytic domains were retrieved from the NCBI’s conserved domain database  was used for heterologous expression  was grown in LB medium with 100 µg/ml ampicillin when needed. Reagents for yeast media were obtained from Difco Laboratories . N-Methyl-1-deoxynojirimycin (NMDNJ) was from Research Chemicals . Enzymes used for DNA procedures were from New England Biolabs , KOD Hot Start DNA Polymerase was from Merck  andpCR2.1-TOPO Vector was from Invitrogen . Unless otherwise stated, all other reagents were from Sigma . UDP-[14C]Glc was synthesized as previously reported with slight modifications (Wright A 1965). Protein concentrations were determined by Bio-Rad Protein Assay as described by the manufacturer.c-myc labeled Ce-uggt-a and Ce-uggt-b optimized versions were synthesized using pREP3X-uggt-a and pREP3X-uggt-b as template   fused to the C-terminal domain of SpUGGT (amino acids 1155 to 1447) named chimera I, and by the N-terminal domain of Ce-UGGT-b (amino acids 1 to1093) fused to the C-terminal domain of SpUGGT (amino acids 1155 to 1447) named chimera II   (constru+-c-myc) . Expressgpt1/alg6S. pombe cells and transformants were selected on MM plates plus adenine containing 15 mM thiamine. To test the accurate expression of the different proteins, 200 µg of S. pombe microsomal proteins were analyzed in 8% SDS-PAGE and subjected to Western blot analysis using an anti-c-myc antibody (Sigma) and a commercial ECL Plus Western Blotting chemiluminescence kit (Thermo Scientific Pierce).Expression plasmids were electroporated into 14C]Glc as a sugar donor and denatured thyroglobulin as a glucosyl acceptor as previously described , and 300 µg of yeast microsomal protein. Reactions were stopped by the addition of 1 ml of 10% trichloroacetic acid. After centrifugation, the pellets were twice washed with 1 ml of 10% trichloroacetic acid and counted. Schizosaccharomyces pombe microsomes were prepared as already described  and structure mapping over sequence alignment were performed with Espript web server (Robert X 2014). Using this alignment and the CtUGGT crystal structure, homology models for Ce-UGGT-a and Ce-UGGT-b were built using Modeler  protein sequences used in phylogenetic inference.Additional File 3 contains the aligned set of 195 UGGT (and UGGT-like) protein sequences used in phylogenetic inference.Additional File 4 shows aminoacidic positions corresponding to each UGGT domain taken as reference sequences.Additional File 5 contains Table S1, Table S2, Table S3 and Supplementary Figure S3.https://itol.embl.de/tree/181461382483101567002659). Supplemental material available at figshare: https://doi.org/10.25387/g3.11234654.The interactive version of the phylogenetic tree has been uploaded to the iTOL server  are lower than that of Ce-UGGTs (0.560). In turn, distances between any of the C. elegans and human HUGTs are comparable to the distance between the two copies of the worm species.Branch lengths in the e-UGGT-a , suggeste-UGGT-a . Table 1Caenorhabditis UGGTs  of recognition and catalytic domains revealed that negative selection prevailed over positive selection in both regions of UGGTs in vertebrates as well as in Caenorhabditis  and UGGT1/UGGT-a as background (or reference). The Relax test revealed significant relaxation of purifying selection in the recognition domain of the Caenorhabditis UGGT-b , that may be obscuring selection relaxation of UGGT-b in relation to UGGT-a. Indeed, when excluding both C. sinica UGGTs from the analysis, significant relaxation in the catalytic domain of UGGT-b is corroborated . Different purifying selection intensities are observed along UGGT-b branches, which depict a more relaxed pattern of evolution in the recognition domain, while the catalytic domain shows a generalized pattern of negative selection intensification, but as mentioned this pattern is reverted when excluding C. sinica . In line with this result, evidence of EDS was found in the recognition domain, but not in the catalytic one as revealed by the BUSTED test (C. sinica UGGTs (P = 0.622).Lineage-based methods were applied to analyze evidence of relaxation or Episodic Diversifying Selection (EDS) in vertebrates and s UGGT-b . In conts UGGT-b . This isTED test , even whSimilar results were obtained among vertebrate UGGT2, where significant relaxation was found in both domains and evidence of EDS only in the recognition domain . While tCaenorhabditis, have been subjected to functional constraints. The catalytic domain is highly conserved as a product of a strong negative selective pressure and shows almost no evidence of positive selection. In contrast, the recognition domain has undergone a positive diversifying selection process in UGGT2/UGGT-b of both lineages. Purifying selective pressure relaxation in the UGGT-b reached higher magnitudes in Caenorhabditis compared to vertebrate UGGT2, especially in its recognition domain , which is in agreement with the higher overall divergence of this paralog in the worm genus.Taken together, these results reflect a generalized background of purifying selection in all UGGTs, suggesting that both duplicates, in vertebrates as well as in alg6gpt1- double mutant S. pombe cells, which lack UGGT and transfer Man9-GlcNAc2 instead of the complete glycan, while UGGT-a was fully active   and thioredoxin-like3 (TRXL3) domains have the same fold belonging to ER luminal chaperones and they have previously been proposed to be involved in the recognition of misfolded proteins  and the other chimeric protein constituted by the N-terminal domain of Ce-UGGT-b fused to the C-terminal domain of SpUGGT was fully inactive (less than 1% of SpUGGT activity).UGGTs are constituted by two highly structurally conserved domains and different chimeras have been constructed combining N-terminal and C-terminal domains from different UGGTs that produced fully active proteins  that gained a new function  in Caenorhabditis.The UDP-glucose:glycoprotein glucosyltransferase gene exists as a single orthologous gene in all major groups of eukaryotes. Only a few protists species that make either very short N-linked glycans or no N-linked glycans at all, lack the uggt gene . On the uggt genes went through independent duplications in Caenorhabditis and vertebrates  (“Wormbase”). Caenorhabditis elegans has fewer interchromosomal gene duplications than expected by chance  sexual chromosome and in the autosome 1 , respectively (“Wormbase”). In H. sapiens, HUGT1 and HUGT2 genes are located in two autosomal chromosomes (the genetic position of HUGT1 is 2q14.3 while that of HUGT2 is 13q32.1)(“http://www.omim.org”). HUGT1, HUGT2, Ce-UGGT-a, and Ce-UGGT-b pairwise distance values were compared, with the lowest score belonging to human paralogs, a compatible result with a HUGT duplication posterior to the nematode-vertebrate common ancestor (NVCA) divergence  selection , acquisition of a new function  or preservation of both genes due to the complementary partitioning of the original function between the two duplicates  . CaenorhHUGTs are differentially regulated. HUGT1 but not HUGT2 is upregulated upon disruption of protein folding in the ER, while the expression of mouse uggt genes is differentially regulated by high concentrations of progesterone (The presence of two UGGT isoforms (HUGT1 and HUGT2) was first reported in humans . NeverthCaenorhabditis  and edem (= ERAD-degradation-enhancing-α-mannosidase-like protein) are also paralogs  interfered worms have only minor phenotypes  was provided by experiments of gene silencing by RNAi. When ire-1uggt-b(RNAi) mutant strain is treated with a very low tunicamycin concentration (that triggers a low level of accumulation of misfolded proteins), worms die or stop their development while no such effect was observed in ire-1uggt-a(RNAi). The lack of Ce-UGGT-b in early development causes general defects that produce larval arrest or cell death  (“Wormbase”). On the other hand, while Ce-UGGT-a is regularly expressed during the entire development, Ce-UGGT-b expression shows a minor peak only at 100 h after fertilization in the mesoderm and ectoderm tissues and in the transition between larva 2 to larva 3 stage (“Wormbase”).Ce-uggt-b homozygous deletion mutant strain is lethal  and proposed that UGGT-b activity was not directly related to the QC cycle   , both seuggt copy in Caenorhabditis and vertebrates lineages, giving rise to different evolutionary pathways of the resulting copies. In vertebrates, UGGT2 may interact with a subset of specific client proteins with minimum or null overlap with those recognized by UGGT1, resulting in the specialization of its function in a background of purifying selection. Within Caenorhabditis, UGGT-b-as evidenced by its sequence evolution and functional assays- has acquired a new role that remains to be characterized, depicting a neofunctionalization process. The independent origin and divergent functions of uggt-2 and uggt-b in both lineages should alert about the improperness of treating them as orthologs.Two independent duplications originated the second


GmC2-58, GmC2-88, and GmC2-148 had higher levels of expression under salt, drought, and abscisic acid (ABA) treatments. GmC2-148, encoding a cell membrane-localized protein, had the highest level of response to various treatments according to real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Under salt and drought stresses, the soybean plants with GmC2-148 transgenic hairy roots showed delayed leaf rolling, a higher content of proline (Pro), and lower contents of H2O2, O2– and malondialdehyde (MDA) compared to those of the empty vector (EV) plants. The results of transgenic Arabidopsis in salt and drought treatments were consistent with those in soybean treatments. In addition, the soybean plants with GmC2-148 transgenic hairy roots increased transcript levels of several abiotic stress-related marker genes, including COR47, NCDE3, NAC11, WRKY13, DREB2A, MYB84, bZIP44, and KIN1 which resulted in enhanced abiotic stress tolerance in soybean. These results indicate that C2 domain genes are involved in response to salt and drought stresses, and this study provides a genome-wide analysis of the C2 domain family in soybean.Plant C2 domain proteins play essential biological functions in numerous plants. In this study, 180 soybean C2 domain genes were identified by screening. Phylogenetic relationship analysis revealed that C2 domain genes fell into three distinct groups with diverged gene structure and conserved functional domain. Chromosomal location analysis indicated that C2 domain genes mapped to 20 chromosomes. The transcript profiles based on RNA-seq data showed that  Environmental stresses caused by such factors as salt and drought significantly affect plant growth and metabolic processes, threaten the survival of plants, and often lead to reduction in yield . In resp2+-dependent membrane targeting domain, this domain shows Ca2+-dependent phospholipid binding activity and a higher sensitivity to salt and drought stresses .In plants, C2 domain genes play important roles, but they vary from species to species . It is rdelivery . Anothert manner . In recet manner , mung bet manner , and Araproteins  were allt stress . In tranol group . Togetheol group . AlthougSoybean is both a major global cereal crop and an essential tool in plant research. Particularly for countries such as China, soybean is a vital economic crop for cooking oil, soy products, soy sauce and protein extracts . Howevercis-elements, and expression patterns in various treatments. To establish the role of the soybean C2 domain genes in response to stresses, we evaluated their response to various treatments, which include salt and drought stresses, as well as ABA treatment. Using real-time quantitative polymerase chain reaction (RT-qPCR) analysis, we revealed a gene, GmC2-148, with the strongest responses to these treatments. We simultaneously verified the resistance of GmC2-148 to abiotic stresses in soybean and Arabidopsis demonstrated that GmC2-148 possibly will improve acceptance to salt and drought stresses. The results presented in this report will expand our knowledge of the C2 domain family in soybean.In this study, we identified 180 soybean C2 domain genes and analyzed their phylogenetic relationship, chromosomal distributions, motif compositions, Arabidopsis were attained from Phytozome1. We downloaded the Hidden Markov model (HMM) profiles of the C2 domain (PF00168) to search C2 domain genes in the soybean dataset using the software HMMER 3.0 (HMMER2) with a threshold of e-value < e–5. The CDD3, Pfam4, and SMART5 databases were used to determine the conservative C2 domain genes. Calculation biophysical properties of C2 domain genes using the online server tool ExPASy6.Protein sequences of the entire C2 domain in soybean and Arabidopsis was performed by ClustalW27. Using pairwise distances and the neighbor-joining (NJ) algorithm, we constructed a phylogenetic tree by MEGA7.0 with 1,000 bootstrap. All C2 domain genes were separately mapped on the 20 soybean chromosomes based on information from the Phytozome database. Multiple collinear scanning toolkits (MCScanX8) with the default parameters were used to analyze the gene replication events.Multiple sequence alignment of the C2 domain genes from soybean and 9. Motif information was obtained by MEME10. Then we imported the images generated by the online websites into TBtools11 to generate a cluster composition  and the cDNA synthesized kit  to obtain cDNA for follow-up experiments. The concentration of cDNA was diluted to about 200 ng/ul.GmC2-58, GmC2-88, and GmC2-148 exposed to the various treatments were analyzed in soybean. RT-qPCR was accompanied with an ABI7500 real-time PCR system  using TransStart Top Green qPCR SuperMix . The data of each gene expression is calculated by using the method of 2−ΔΔCt method. Expression of GmC2-58, GmC2-88, and GmC2-148 were measured relative to the control (0 h). Using actin gene as internal control, the template cDNA was normalized. The total volume containing 2 × Taq PCR Master Mix was 20 μl . The primer sequences used in this paper are all shown in the The expression levels of three C2 domain genes, 14 to annotate and analyze the C2 domain family.According to GmC2-148 was specifically amplified by PCR, then the correctly sequenced plasmid was amplified with the splice primer with GFP, and transformed in Escherichia coli to extract the fusion plasmid of GmC2-148-GFP. The recombinant plasmid of GmC2-148-GFP was transformed into Arabidopsis protoplasts using PEG4000-mediated method (We constructed an expression vector with green fluorescent protein (GFP) marker for subsequent subcellular localization analysis as described earlier . The codd method . After iGmC2-148 transgenic hairy roots, the GmC2-148-3301 recombinant vector and empty vector (EV) were introduced into Agrobacterium competent cells of strain K599 and then transformed into soybean hairy roots by A. rhizogenes-mediated transformation as described earlier  and GmC2-148 transgenic plants were treated with 250 mM NaCl solution for 7 days. During the drought treatment, Arabidopsis were grown while watering was withheld for 10 days. In the Arabidopsis root length experiment, we used 6 and 9% (w/v) polyethylene glycol (PEG) to simulate drought, and treated Arabidopsis with 90 and 110 mM NaCl, 10 and 12 μM ABA to observe the root length phenotype.As for experiment on salt treatment of 2O2, and O2– contents from soybean leaves after the salt and drought stress treatments. For each physiological index, we sampled leaves from three individuals. Contents of MDA, Pro, H2O2, and O2– were measured 0.1 g from leaf samples follow the instructions of the kit . Determination of absorbance value by Varioskan LUX multimode enzyme labeling instrument .During this study, we measured malondialdehyde (MDA), Pro, HTo determine the content of chlorophyll, took 0.1 g from soybean leaves and cut them up, placed in a centrifuge tube, and filled up to 10 ml with 80% acetone solution before placing the tube in the dark for 24 h. The chlorophyll extract’s reflectance was then measured at wavelengths of 665 and 649 nm to determine the chlorophyll content. An 80% acetone solution was used as the control.R1/R2 × 100%.To prepare materials for measurement of electrical conductivity, plant leaves of the same size were cleaned with paper and any water remaining on their surfaces was absorbed with filter paper. Then we cut the leaves into pieces of the same size, mixed them evenly and took 0.1 g of the cut pieces to soak them in 10 ml water for 12 h. Next, we measured the conductivity R1 of the solution of steeped leaf sample with a conductivity meter then putting samples in boiling water for 30 min and then cooling them to 25°C. We then measured R2 to calculate relative conductivity of samples based on the following equation: Individual intact leaf samples from salt and drought stressed soybean and a control soybean group of plants were used in two staining procedures to observe the degree of damage to the leaves. For Nitrogen Blue Tetrazole (NBT) staining, the cut leaves were soaked in NBT solution for 14 h, and then immersed in 75% ethanol for decoloration until the sample became transparent. For trypan blue staining, we first prepared 10 ml of 0.4% trypan blue solution that was filtered and sterilized before adding 90 ml 1 × PBS solution. The leaves were placed in 100 ml of the mixed solution and kept at 37°C in the dark for 24 h. Dyed leaves were then added to a 1:3 ethanol:glacial acetic acid solution to decolorize leaves at 37°C in the dark for 24 h until the samples became transparent.Relative water content was determined using the following equation: relative water content (%) = (fresh weight − dry weight)/(saturated fresh weight − dry weight) × 100%. Soybean leaves were cut into pieces and then weighed to obtain fresh weight. They were subsequently soaked for a few hours until the leaves were saturated with water. Then their leaf surfaces were dried with absorbent paper and the leaves were weighed immediately to obtain their saturated fresh weight. The same leaves were then oven-dried at about 70–80°C for 2 days until they reached a constant weight. Dried samples were placed them on a table at 25°C for 2 h before measuring the dry weight .G. max Wm82.a2.v1). After screening from SMART and Pfam databases, we identified 180 C2 domain family members in soybean. The statistical results exhibited that the amino acid sequence length of the 180 C2 domain genes varied from the shortest of 128 amino acids to the longest of 1,181 amino acids, and the length of CDS varied from 384 to 3,543. The isoelectric point (pI) varied from 4.8 to 5.32, and the molecular weights (MW) ranged from 27.9 to 292.5 kDa  and six subgroups  according to their genetic relationships  residues (2+ plays a vital role as a second messenger in response to abiotic stresses (2+ is induced by salt and high osmotic pressure, and the elevated level is connected to the expression of stress-responsive genes (2+ signaling (Arabidopsis C2 domain protein, involved in response to freeze stress with Ca2+-dependent manner (GmC2-148 and Ca2+ needs further elaboration.In plants, the C2 domain proteins are reportedly involved in diverse processes, including abiotic stress, pollen fertility, and membrane targeting and leaf senescence in a Cat manner . Previouresidues . As repostresses . Elevative genes . Throughignaling . The SYTt manner . In thisGmC2-148, encoding a cell membrane-localized protein, is a positive regulator of plant tolerance to salt and drought stresses by activating abiotic stress-related genes. However, further study is needed to determine the specific functions of the C2 domain genes. To sum up, the present study investigates the importance of C2 domain genes in abiotic stress-tolerance in soybean.In the present study, we implemented a genome-wide analysis of the C2 domain gene family in soybean. A total of 180 C2 domain genes were identified based on the conserved C2 domain, and the genes were classified into three major groups, I, II, and III. Most members of group I responded to salt, drought and ABA treatments. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Z-SX coordinated the project, conceived and designed the experiments, and edited the manuscript. YS performed the experiments and wrote the first draft. J-YZ revised the manuscript. Y-TL and P-GZ contributed to the data analysis and managed reagents. Z-WF contributed with the valuable discussions. JG put forward valuable opinions when revising the manuscript. All authors reviewed and approved the final 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.


The Target of Rapamycin (TOR) is a highly conserved serine/threonine protein kinase that performs essential roles in the control of cellular growth and metabolism. TOR acts in two distinct multiprotein complexes, TORC1 and TORC2 (mTORC1 and mTORC2 in humans), which maintain different aspects of cellular homeostasis and orchestrate the cellular responses to diverse environmental challenges. Interest in understanding TOR signaling is further motivated by observations that link aberrant TOR signaling to a variety of diseases, ranging from epilepsy to cancer. In the last few years, driven in large part by recent advances in cryo-electron microscopy, there has been an explosion of available structures of (m)TORC1 and its regulators, as well as several (m)TORC2 structures, derived from both yeast and mammals. In this review, we highlight and summarize the main findings from these reports and discuss both the fascinating and unexpected molecular biology revealed and how this knowledge will potentially contribute to new therapeutic strategies to manipulate signaling through these clinically relevant pathways. TOR (Target Of Rapamycin) genes were first identified in a screen of yeast mutants that were resistant to the macrolide rapamycin ,3231,32]Once recruited to the lysosomal membrane, subsequent activation of mTORC1 requires interaction with the other major GTPase regulator of mTORC1, Rheb. Unlike the Rags, Rheb can interact directly with the lysosomal membrane via its farnesylated C-terminal tail . In metaTogether, the structures described above suggest a model for the anchoring of the activated mTORC1 complex to the lysosomal membrane D 31,32],3231,32]Binding of Rheb to mTOR requires a significant movement of the N-HEAT domain towards the M-HEAT domain A. This dThe conformational changes observed upon mTORC1 activation also give insights into the effects of particular mutations. Hyperactivating and recurrent mutations in mTOR that have been associated with cancer appear to cluster to the FAT and KD D, and soThe Rag GTPases have a very low intrinsic activity , which iCompared to nucleotide exchange, the action of the dedicated RagA/B GAP, named GAP activity toward Rags (GATOR1), is better defined but also still only partially characterized  Figure A. GATOR1In a similar vein, the RagC/D nucleotide-loading status is also controlled by a dedicated GAP, the folliculin (FLCN)-folliculin interacting protein (FNIP1/2) complex . The FLCComparison of the conformation of the RagA/RagC G domains also reveals different degrees of opening of the inter G-domain space, with the FLCN-FNIP2 bound structure presenting a much wider displacement of the G-domains compared to when either GATOR1 or Raptor is bound D. IntersChaetomium thermophilum GAP domain solved . T. T99]. TSubstrate recruitment to mTORC2/TORC2 appears to be mediated by mSin1/Avo1 via its conserved region in the middle (CRIM) domain ,103. In Finally, the recent structure of mTORC2 also revealed two small molecule binding sites: the “A-site” (that binds ATP) located in Rictor and the “I-site”  located in the mTOR FAT domain . The A-sAs mentioned previously, mTOR is the founding member of the PIKK family. These proteins were initially misclassified as PIKs but now have emerged as an important protein family with a wide range of functions in the cell. There are five known PIKKs in addition to mTOR: DNA-PKcs, ATR and ATM (involved in DNA damage repair), SMG1 (involved in mRNA decay) and TRRAP (involved in chromatin remodeling) . All PIKKluyveromyces marxianus (KmTor) [The two-lobed PIKK KD presents several conserved elements: the ATP loop in the N-lobe, and the catalytic and activation loops, as well as the PIKK-regulatory domain (PRD), in the C-lobe  Figure B. The PR (KmTor)  and is d (KmTor) , althoug (KmTor) . Similar (KmTor)   . Accordi (KmTor) . TogetheThe PIKKs SMG1, Tra1 and DNA-PKcs (but not ATM and ATR) each have an alpha-helical bundle in the KD N-lobe, reminiscent of the FRB domain of mTOR. However, the relative positioning of this bundle in Tra1 is tilted towards the equivalent of the LBE domain in the C-lobe, which occludes the putative active site. This, coupled with the lack of conservation of the catalytic residues, likely explains the lack of kinase activity of Tra1/TRRAP . HoweverThe cellular assembly of multi-subunit protein complexes is a major feat that requires the concerted action of chaperones and proteins that aid in both proper subunit folding and inter-subunit interactions. This is particularly important for PIKKs, given their complex topology. Tel2 was identified as an essential protein needed for PIKK stabilization in vivo . Tel2 biBeyond mTOR, proper folding of the other mTORC1 subunits is also required. Recently, it has been shown that both mLst8 and Raptor bind to the chaperonin CCT via their β-propeller/WD40 domains, an interaction which is required for proper folding and mTORC1 assembly . This inThe structural information presented in this review has significantly increased our knowledge of the molecular mechanisms by which mTOR activity is controlled. In particular, the structures of multiple PIKKs have revealed that findings for one member of the family can help in understanding of the regulation and function of other members of the family. However, many questions remain. For example, structures of many important TORC1 regulators including GATOR2, SAMTOR and KICSTOR remain to be reported, as well as regulators of TORC2 . In a siDysregulation of mTORC1 , mTORC2


One of the applications is to use as a high-throughput screening method for pathogenicity of genomes. Unlike sequence homology methods, protein comparison at a functional level provides us with a unique opportunity to classify proteins, based on their functional structures without dealing with sequence complexity of distantly related species. Protein functions can be abstractly described by a set of protein functional domains, such as PfamA domains; a set of genomes can then be mapped to a matrix, with each row representing a genome, and the columns representing the presence or absence of a given functional domain. However, a powerful tool is needed to analyze the large sparse matrices generated by millions of genomes that will become available in the near future. The ProdMX is a tool with user-friendly utilities developed to facilitate high-throughput analysis of proteins with an ability to be included as an effective module in the high-throughput pipeline. The ProdMX employs a compressed sparse matrix algorithm to reduce computational resources and time used to perform the matrix manipulation during functional domain analysis. The ProdMX is a free and publicly available Python package which can be installed with popular package mangers such as PyPI and Conda, or with a standard installer from source code available on the ProdMX GitHub repository at  Such tool requires a critical feature that allow users to quickly customize sets of interesting functions or organisms based on problem sets. Early tools for domain architecture comparison, such as CDART Here, we present the ProdMX tool, a standalone Python tool that empowers researchers to explore functional domains and domain architectures of proteins across genomes of interest. With the state-of-the-art matrix compression algorithm, the ProdMX can be applied in the variety of applications including the high throughput screening for pathogenicity of genomes. The tool aims to reduce time and the computational resource used to calculate a large matrix of all-to-all comparison of functional domains or domain architectures. The ProdMX enables researchers familiar with the command prompt or the Python programming language to rapidly analyze specificity or conservation of functional domains or domain architectures. The ProdMX accelerates the protein functional research by offering an intuitive tool that can handle a massive amount of proteins when the computational resource is limited.22.1The ProdMX tool was designed to handle the large-scale analysis of both functional domains and domain architectures across million genomes. Due to the nature of the high complexity in the arrangement of protein functional domains in various genomes, the resulting matrix is both massive  and sparse . The Compress Sparse Row (CSR) algorithm The data manipulation in the tool was handled with the Pandas package For the analysis and report generating parts, we designed the ProdMX tool to work as a Python package. This design allows users to flexibly use the ProdMX tool in the chain of commands with other tools in the user’s pipelines. In addition to this package design, users can quickly test the prototype of code or perform analyses on Jupyter Notebook, Python web-based interactive development environment 2.2Escherichia coli sequences, not derived from single cell nor metagenome project, having 0.8 or more for total and sequence quality scores in the dBBQs database E. coli genomes . These sets of genomes were also implemented in the database with a sparse structure in SQLite for retrieval speed comparison. We examined the average CPU runtime for calculating core functional domains and core domain architectures of 100 replicates for each set of genomes using a calCore utitity in ProdMX and SQLite queries .33.170 protein (RpoD) E. coli genomes from the previous section  allows us to store raw results of protein ids and their domains for further analysis. The example of command for building the sparse matrix folder in the command-line environment using the test data of high-quality prodmx-buildDomain -i input_ecoli_id_hmm.tsv -o domain_matrix_fol -kThe ProdMX tool provides analysis utilities in the Python package for regular use and the interactive Python environment. In this demonstration, we choose to analyze our protein functional domain in Jupyter Notebook, the popular interactive Python environment. The binary matrix of presence and absence of each functional domain for each genome is then loaded to the memory by following commands:import prodmximport pandas as pdbinary_matrix = prodmx.loadBinMatrix(matrix_fol='domain_matrix_fol')70 proteins (PF04542) is present in core functional domains (95% or more in conservation) across our set of E. coli genomes or not, we need to supply the calCore function with the information of list of genome labels and list of available protein functional domains as follows:Further, to check if the region 2 domain of σdf_core_domain = binary_matrix.calCoreE. coli genome possessing the region 2 domain of σ70 (PF04542) can be retrieved by following command:Using a Pandas DataFrame command, we can select rows based on condition. The number of df_core_domain[df_core_domain['col_name'] == 'PF04542′]70 for each genome to the output file in the tab-delimited format :Finally, the command below will be used to write all protein ids associated with the region 2 domain of σbinary_matrix.getProteinId3.23.2.1Clostridioides difficile or formerly known as Clostridium difficile is a group of bacteria that cause severe damage to the colon with diarrhea symptoms. This gram-positive species is one of the most common bacteria found in healthcare-associated infections (HAIs) in the United States C. difficile organize by two toxin genes TcdA and TcdBTcdR TcdA and TcdB protein. The dictionary between protein name and domain architectures were created as follows.To check for exotoxin genes, we retrieved the domain architectures from UniProt dict_tox = {'tcdB':'PF12918_PF12919_PF11713_PF12920_PF19127_PF19127′, 'tcdA':'PF12918_PF12919_PF11713_PF12920_PF19127_PF19127_PF19127_PF19127_PF19127′}Using Pandas DataFame, we can create the data table for the in silico-screening of exotoxin in the unknown genomes by the code following:list_result = for genome_id in count_matrix.getRow:x = count_matrix.sumColumn, dict_tox.get('tcdA')])['col_sum'].tolistlist_result.append([genome_id]+x)header=pd.DataFrameThe table of genome and protein ids associating with exotoxins can be retrieved as follows:count_matrix.getProteinId, dict_tox.get('tcdA')], output='clostridiales_exotoxin_protein_id.txt')3.2.2Clostridium botulinum can cause the disease botulism which is a potentially fatal disease in human C. botulinum comprise of ntnh and bont genes with the alternative sigma factor botR to regulate the expression (The botulinum neurotoxins (BoNTs) produced by the strains of pression B. Referrdict_tox = {'bont': 'PF01742_PF07952_PF07953_PF07951′}count_matrix.getProteinId, output='clostridiales_neurotoxin_protein_id.txt')https://github.com/visanuwan/prodmx).The test data and extended versions for example 3.1 and 3.2 in Jupyter Notebook can be downloaded at the ProdMX GitHub repository (4Here we introduce the ProdMX tool, which provides a native Python environment for analysis of the protein functional domain. While the functional domain analysis can be performed with the web-based applications, analyses of large queries are often limited by the bandwidth required to transfer data over the internet. Analyses with standalone tools are usually found to have insufficient memory issues due to the management of enormous sparse matrices and the complexity of dependencies required for tool installations. Overall results of benchmarking from ProdMX showed remarkably better performance over SQLite with sparse matrix schema implementation. With the provided use cases, ProdMX can be used as an effective tool for the high-throughput screening. We expect that the ProdMX tool will aid the scientific community in performing large queries and accelerate comparative genomics research that relies on accurate and clade-specific measures of protein functions.https://app.box.com/s/n7chra1me0g5nv6yclzmshzns0drvxzo.Publicly available datasets were analyzed in this study. The data for benchmarking, list of genome assembly accessions, and HMMER results associated with the genomes from GenBank are available at College of Medicine and the Department of Biomedical Informatics at UAMS, the Helen Adams & Arkansas Research Alliance Endowment. The National Science Foundation under Award No. OIA-1946391. National Institute of General Medical Sciences of the National Institutes of Health [P20GM125503] awarded to I.N.This research was funded in part by the Visanu Wanchai: Conceptualization, Software, Visualization, Writing - original draft, Writing - review & editing. Intawat Nookaew: Visualization, Validation, Writing - review & editing, Supervision. David W. Ussery: Supervision, Validation, Resources, Writing - review & editing.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.