The current burst of device concepts based on nanoscale domain-control in magnetically and electrically ordered systems motivates us to review the recent development in the design of domain engineered oxide heterostructures. The improved ability to design and control advanced ferroic domain architectures came hand in hand with major advances in investigation capacity of nanoscale ferroic states. The new avenues offered by prototypical multiferroic materials, in which electric and magnetic orders coexist, are expanding beyond the canonical low-energy-consuming electrical control of a net magnetization. Domain pattern inversion, for instance, holds promises of increased functionalities. In this review, we first describe the recent development in the creation of controlled ferroelectric and multiferroic domain architectures in thin films and multilayers. We then present techniques for probing the domain state with a particular focus on non-invasive tools allowing the determination of buried ferroic states. Finally, we discuss the switching events and their domain analysis, providing critical insight into the evolution of device concepts involving multiferroic thin films and heterostructures. Multiferroic materials with coexisting electric and magnetic order are technologically attractive ,2,3,4. IDomain state and domain wall engineering are essential for applications of ferroic thin films since the switching mechanism is driven by nucleation and motion of domain walls. In thin films hosting a reversible electric polarization, the interplay between epitaxial strain and electrostatics renders the control of the domain architecture possible using the lattice and charge degrees of freedom . OptimalThe increased control of complex domain architecture using charged surface states at oxide interfaces, defects in stoichiometry or domain imprint across interfaces, as described in this review, leads to new developments for buried domain states in oxide heterostructures. Here, we place emphasis on the recent advances in domain design and manipulation in ferroelectric and multiferroic thin films and heterostructures. Therefore, the first part of this review is devoted to ferroic domain design in thin layers and superlattices. In the following sections, we review the progress and challenges in accessing the domain state and the switching events in multiferroics, especially in buried layers.The domain state in ferroelectric thin films is set by a combination of factors such as strain, chemistry and charge screening at the interfaces, and layer thickness. Domain engineering in ferroelectric thin films using epitaxial strain has been reviewed in great detail, see ,27,28 foThe onset of net ferroelectric polarization triggers the accumulation of bound surface charges in ferroelectric materials. This charge accumulation, in return, creates a depolarizing field oriented oppositely to the ferroelectric polarization. The depolarizing field plays a decisive role in determining the domain state and can, for instance, induce domain splitting ,35 or, iBy acting on the screening of ferroelectric bound charges, the depolarizing field strength can be modulated. The electrostatic environment can be engineered by interfacing ferroelectric layers with metallic/dielectric layers or by changing the environment, i.e., adsorbates and surrounding gas atmosphere. This represents alternative routes towards control of polarization state and domain formation in ferroelectric thin films.0.2Ti0.8)O3 (PZT), charges accumulate mainly at top and bottom interfaces. The introduction of a metallic buffer provides sufficient charge screening to stabilize a single-domain state. However, intentionally introducing a large depolarizing field can benefit the ferroelectric properties as demonstrated by Liu et al. 2O4 (CFO)3 (LSMO) . A key r3 (LSMO) .90Fe10 (CoFe)/BFO heterostructure by imaging magnetization using scanning electron microscopy with polarization analysis (SEMPA) and underlying polarization with back-scattered electrons (BSE) (3 [3 [(ii) In exchange-coupled multiferroic composites, the interaction occurs between a ferromagnet and a single-phase multiferroic magnetoelectric with uncompensated antiferromagnetic order. The electric field acts on both ferroelectric polarization and the direction of antiferromagnetic spin ordering in the multiferroic. This allows a direct exchange effect between the antiferromagnetic and ferromagnetic spin orderings. Although the physics of exchange coupling is different from that of strain coupling, both produce lateral modulations of magnetic anisotropy leading to domain transfer. Trassin et al.  determinns (BSE) f,g. A on(BSE) (3  and LuMnE)  [(iii) Charge-coupled artificial multiferroics make use of the ferroelectric field effect: Bound charges at the ferroelectric interface are screened by the ferromagnetic layer, leading to either accumulation (hole-doped) or depletion (electron-doped) states in the ferromagnet when the ferroelectric polarization is pointing away from or towards the ferromagnet, respectively. If the ferromagnet is a strongly correlated system, this can result in drastic alterations of magnetization and even domain imprint. This mechanism is different from the two mentioned previously, because changes in magnetization are limited to interfaces, up to a screening length . However3 (SRO) .Domain investigation is a critical element in the understanding of complex oxide multifunctional layers  since thBy design, the ferroelectric component in artificial multiferroic heterostructures is covered by a conducting ferromagnetic layer. Non-crystalline, oxidation-sensitive ferromagnetic layers need to be grown on top of a ferroelectric layer, after the high-temperature ferroelectric material deposition process. In the case of crystalline ferromagnetic layers for strain-induced interfacial coupling, the ferroelectric film plays the role of the substrate to influence the ferromagnetic lattice and therefore lies underneath the metallic layer. This buried nature results in a loss of information about the ferroelectric domain architecture when using conventional techniques such as PFM. The magnetic domain state of the top layer can, however, be accessed directly with scanning probe microscopy, see 3/STO (PTO/STO) superlattices . This phenomenon concerns the entire switched volume. The chemical profiles of various elements are mapped after a local switching event in Another approach towards the determination of buried polar states consists of probing the surface chemistry of ferroelectric films. Polarization switching events have mostly been analyzed with the assumption of unchanged stoichiometry. Ievlev et al.  revealedWith the increasing knowledge of the impact of surface chemistry on switching properties of ferroelectric materials, these works ,105 furtNanoscale domain patterns in ferroelectric thin films are most commonly imaged by PFM . HoweverX-ray diffraction of thin films is an effective non-invasive probe of ferroelectric polarization via measurement of structural parameters. In single layers, tetragonality and thus indirectly polarization can be accessed through measurements of the out-of-plane and in-plane lattice parameters. However, detection in the ultrathin regime is limited by the reduced sample volume. The superlattice architecture is, therefore, used to reproduce ultrathin film behavior while providing increased active volume. The ferroelectric/dielectric superlattices became the model system for such analysis ,110. It 3 thin films [Hadjimichael et al.  recentlyin films  further in films  and polain films .Optical SHG is a non-destructive, non-invasive probing technique for polarization in ultrathin ferroelectric films. The SHG technique is sensitive to inversion symmetry breaking and, therefore. ideal for probing ferroelectricity. Previously devoted to bulk ferroic materials investigation, SHG has become an essential tool for the examination of ferroelectric domains in thin films. Its potential as a ferroic state probe has been the topic of several reviews dealing with bulk and thin film materials ,124,125.Despite the lack of spatial resolution , a net polarization can be optically detected in domain-engineered films, exhibiting either a single-domain state or a domain architecture leading to a net polarization. The polarization analysis using SHG is not affected by increasing leakage currents in the low thickness range, which prevent the determination of intrinsic ferroelectric behavior using the conventional ferroelectric testing approach. The SHG investigation, therefore, enables the determination at the ultrathin limit of ferroelectricity.Recently, a specific non-linear optical signature of tilted 180° ferroelectric domain walls corresponding to a mixed Ising–Néel domain-wall type was shown for the first time b–d 99,1,199,126.3), the weak ferromagnetic behavior has been directly observed using MFM at 50 K, see Magnetic domain pattern investigation at the nanoscale is nowadays enabled by the development of magnetic force microscopy (MFM) operating under magnetic fields and cryogenic temperatures. The developments in low-temperature scanning probe microscopy led to major improvements in measurement sensitivity ,130,131.The single-spin magnetometer technique is currently pushing the minimum detectable magnetic moment to even lower values (a few femtotesla). It is based on a point-like impurity nitrogen-vacancy (NV) defect in diamond ,137,138 2O3 magnetoelectric crystal using optical SHG [3 crystals [In some cases, SHG is sensitive to the reduction of symmetry through the magnetic ordering of spins. In a seminal demonstration of this concept, antiferromagnetic domains were imaged in Crical SHG . More reical SHG . Furthercrystals .As described above, the domain analysis post switching provides critical insight into the ferroic behavior. Efforts are now focusing on operando measurements, i.e., probing evolving magnetic and electric domain states during the application of an external field. The investigation of multiferroic switching dynamics, involving domain wall motion, is expected to lead to discoveries beyond the determination of the switching time-scale. The investigation of artificial multiferroic systems is accompanied by the challenge of observing a buried switching event operando.In ferroic materials, beyond the iconic square-like hysteresis of the macroscopic response subject to an applied conjugate field, an understanding and control of ferroic order at the domain level is highly desired. A magnetoelectric multiferroic switch can express itself as a change of electric (magnetic) domain state under the application of a magnetic (electric) field in the remanent state. Magnetoelectric behavior can be demonstrated at the scale of a single ferroic domain and domain walls. The few existing measurements on dynamics of magnetoelectric switching are based on optical SHG and PFM imaging with SHG having the advantage of operando probing the domain state locally during the magnetoelectric switch. 3, SHG imaging revealed the domain architecture during the polarization flop induced by magnetic field [ic field . The spaIn a multiferroic solid solution between lead zirconium titanate (PZT) and lead iron tantalate (PFT), PZTFT, PFM measurements showed a change in the ferroelectric domain population under an application of a magnetic field .4 single crystals [The desired energy-efficient control of magnetic order by electric field is shown in crystals . The magcrystals ,141 -oriented orthorhombic substrate results in a stripe-like domain pattern . This anisotropic domain architecture imposes periodic electrostatic and elastic boundary conditions at each domain wall which can preserve the memory of the initial domain pattern, and hence the ferroelectric (multiferroic) domain state. The magnetization reversal induced by an electric field in multiferroic BFO thin films relies precisely on such a memory effect of the domain pattern. For a given range of pulse widths and electric field amplitudes, a switching event occurs within each domain. The combination of elastic and electrostatic boundary conditions results in an unchanged domain pattern after local polarization rotation  or reverThis further suggests strain engineering as a possible route towards the inversion of domain pattern in a wide variety of ferroic systems.Artificial multiferroic heterostructures hold promises for oxide electronics with low-power consumption at room temperature ,64. OperIn thin-film artificial multiferroic heterostructures, the nanoscale ferroelectric domain architecture cannot be optically resolved. Understanding the dynamics of magnetoelectric poling is, however, crucial for any technological implementations. De Luca et al.  shed somThe experimental access to ferroic domain states is in ongoing progress. New concepts such as dynamical multiferroicity, i.e., generation of magnetization from varying electric polarization, could enable the non-invasive probe of domain wall movement using NV center magnetometry ,148. TheFurthermore, the emergence of polarization and related electrostatic effects can be visualized during deposition ,153,154 The domain visualization during the film deposition remains, however, a challenge. Improving spatial resolution in non-invasive probes would bring an understanding of multiferroic domain formation during the synthesis process. Moreover, recent works have shown that ferroelectric domain walls and multiferroic states can be deterministically tuned by optical means ,156,157.


Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including energy metabolism, calcium homeostasis and apoptosis. Miro proteins (Miros) are “atypical” Ras superfamily GTPases that display unique domain architecture and subcellular localisation regulating mitochondrial transport, autophagy and calcium sensing. Here, we present systematic catalytic domain characterisation and structural analyses of human Miros. Despite lacking key conserved catalytic residues , the Miro N-terminal GTPase domains display GTPase activity. Surprisingly, the C-terminal GTPase domains previously assumed to be “relic” domains were also active. Moreover, Miros show substrate promiscuity and function as NTPases. Molecular docking and structural analyses of Miros revealed unusual features in the Switch I and II regions, facilitating promiscuous substrate binding and suggesting the usage of a novel hydrolytic mechanism. The key substitution in position 13 in the Miros leads us to suggest the existence of an “internal arginine finger”, allowing an unusual catalytic mechanism that does not require GAP protein. Together, the data presented here indicate novel catalytic functions of human Miro atypical GTPases through altered catalytic mechanisms. Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including eukaryotic energy metabolism and cell survival ,2. The mAmongst the many cellular GTPases there exist two isoforms of “atypical” Rho GTPases, named mitochondrial Rhos (Miro), have been established to play critical roles in mitochondrial transport, morphology, and homeostasis ,7. MitocDrosophila and TRAK1/2 in humans respectively [The Miro proteins are conserved across eukaryotic species , and mouectively . When inectively .Xenopus embryos [In addition, further studies on the molecular functions of Miro proteins revealed their role in developmental processes, neurological disorders, cell migration and susceptibility to microbial infections ,12,13. R embryos . Moreove embryos ,16. A Mi embryos ,17,18. Drosophila or vertebrate Miros have revealed a conserved role for Miro proteins in the determination of mitochondrial size, distribution and transport across the species [Drosophila Miro (dMiro) (GV13), which contains a mutation in its N-terminal GTPase domain equivalent to the well-established and analogous G12V constitutively active Ras mutant, showed neomorphic phenotypic effects that are reported to be unrelated to GTPase function [Studies on the N-terminal GTPase domain of  species ,19. Loss species . In agreThe exclusive cellular role of the Miro CT-GTPase domain came to light following the analysis of the formation of the endoplasmic reticulum mitochondria encounter structure (ERMES) complex, which tethers the ER to the mitochondrion . The forin-vitro characterisation of the catalytic activity of the GTPase domains of the two human Miro proteins. The N-terminal GTPase domain displays GTPase activity despite the absence of key Ras residues including Y32, T35, G60 and Q61. Surprisingly, when the catalytic ability of the previously reported “relic” C-terminal GTPase domains of Miro1 and Miro2 was analysed, they were seen to be active. Moreover, they displayed substrate promiscuity, with hydrolytic activity against ATP and UTP observed in addition to GTP, demonstrating that these domains function as NTPases rather than archetypal GTPases. Furthermore, structural modelling and analysis revealed the Miro proteins have unusual structural features that may function as an ‘internal arginine finger’ that enables unusual transition state and nucleotide hydrolysis. Together, the data presented here shows the novel catalytic functions of this atypical GTPase through altered catalytic mechanisms.Here, we performed an 2+ binding, guanine nucleotide binding and hydrolysis [2, which acts as a co-factor.The primary sequences of the GTPase domains of both Miro1 and Miro2 resemble the G domains of classical Ras superfamily proteins B. For thdrolysis ,23, showdrolysis ,19, we s2, as well as both CaCl2 and MgCl2. The results showed that there was no selective preference for either of the cations  A. LikewiInterestingly, the N-terminal and C-terminal catalytic domains of the human Miro proteins show similar enzymatic hydrolysis in end point assays when tested against GTP. The intracellular concentrations of nucleotides rank in the order of ATP > GTP > UTP > CTP . It is tThe two important regions of the Ras GTPases that permit these molecules to function as molecular switches are the Switch I  and Switch II . While the Switch I region is involved in interactions with downstream effectors and stabilisation of the nucleotide γ-phosphate, the Switch II region participates in nucleotide hydrolysis. The conformational change and stabilisation of these two regions overall dictates the activity of the enzyme, effector binding and nucleotide exchange . The impAlthough both the N- and C-terminal GTPase domains of the Miro proteins display hydrolytic activity towards GTP, examination of their sequences reveals a striking lack of conservation in the aforementioned residues , which had led to them being dismissed as relic domains  Figure B. To recIn the N-terminal GTPase domains of Miro 1 and Miro 2, the residues equivalent to Ras Y32, T35, G60 and Q61 are not conserved and have the following mutations in both the Miro1 and Miro2 NT-GTPase domains: Y32V, T35R, G60E and Q61A B. In ourResidue T35 is known to make contacts with the γ-phosphate in the canonical GTPases and its mutation to proline or alanine results in a drastic reduction in nucleotide association capacity . The T35In the Switch II region, the Miro N-terminal GTPase domains display conservation of the aspartate in the DTAGQ Switch II motif but all the other residues are significantly altered B, resultSimilar to the N-terminal domain, the C-terminal domains of Miro1 and 2 display a completely different pattern of residues to those conserved in the Ras-like GTPase enzymes. Here, Y32, T35, G60 and Q61 are present as K, S, E, S and D, P, D, T in Miro1 and 2 respectively B. To assIn addition to these observations, another point of interest in the Miro1 C-terminal GTPase structure is the lysine present at position 12 D. MutatiComparison with other atypical GTPases, Centaurin γ1, RND1 and RND3, reveals that within the common fold, the largest structural difference can be seen in loop G2/Switch I . Here, tThe biochemical assays have established the hydrolytic activity of the Miro GTPase domains, but the sequence and structural analysis show clearly that the Miro GTPases cannot utilize the canonical hydrolysis mechanism found in other GTPases, or, indeed, the mechanisms of the atypical GTPase enzymes. Therefore, the mechanism through which hydrolysis occurs remains to be characterised. In the Miro1 N-terminal GTPase model, two arginine residues, one in position 35, the other at position 13, flank the terminal phosphate groups of the nucleotide, with R13 appearing to occupy a similar position to the arginine finger of the RasGAP p120  before transformation into chemically competent TOP10 E. coli C41 cells freshly transformed with either the Miro N-terminal/C-terminal GTPase domains or the EF-CT-GTPase construct were used for starter cultures that was then used to inoculate large 2-6 litre cultures. Miro protein overexpression was induced using 1 mM IPTG at 18 °C for the N- and C-terminal GTPase constructs and 0.1 mM IPTG for the longer EF-CT-GTPase constructs overnight. Cells were lysed and cell-free supernatant obtained by centrifugation of the solution at 18000× g, 4 °C for 1 h. The soluble fraction was then passed through a Ni-NTA column attached to an ÄKTA Prime fast protein liquid chromatography (FPLC) system. The separation was achieved by subjecting the sample to a gradient of imidazole from 50–500 mM in a buffer containing 50 mM HEPES, 500 mM NaCl, 5% glycerol. Fractions containing the proteins of interest were identified by SDS-PAGE analysis, and were pooled and concentrated using Amicon 10 kDa cutoff filters. Proteins were then loaded on to a 16/60 S200 gel filtration column equilibrated in a running buffer containing 25 mM HEPES, 300 mM NaCl, 2% glycerol, 5 mM DTT. The purified recombinant human Miro proteins were then used for further characterisation.2, CaCl2 or both cations) and assay buffer . After 30 minutes, the reaction was stopped by addition of the “PiColourLock™ Gold Mix”. This plate was shaken gently and incubated for 5 minutes at room temperature prior to addition of the PiColourLock™ stabiliser compound. After 15 minutes, the absorbance of each well was read at 635 nm on a FLUOStar Omega plate reader. Absorbance values were generated by subtracting blank absorbance obtained from test reaction mix containing all components but Miro enzyme from each raw value. Every experiment variant was repeated 3–5 times to determine average release of phosphate from the nucleotides at 25 degrees., and mean values used to generate bar charts, with standard error of the mean calculated for each experiment. PiColourLock™ Gold, a modified form of the standard malachite green assay kit  was utilised as a means of verifying the hydrolytic capacity of target enzymes. A standard curve for phosphate concentration with this kit was generated based on the manufacturer’s instructions. This standard curve was used to assess phosphate generation by target enzymes when assays were performed according to the standard operating procedure supplied by the manufacturer. A typical reaction mixture comprised the following: the enzyme of choice, GTP and one of more cations (2 mM each of MgCl3 box centred on the 3′ hydroxyl group of the bound GDP. Models were initially analysed in UCSD Chimera [Miro1 and 2 N-terminal domain models were produced through input of the amino acid sequence of residues 7–169 of Miro1 and residues 3–169 of Miro2 into the Phyre2 webserver , followe Chimera  were com Chimera  for gene Chimera  and ESPr Chimera  webserve


Ferroic materials are well known to exhibit heterogeneity in the form of domain walls. Understanding the properties of these boundaries is crucial for controlling functionality with external stimuli and for realizing their potential for ultra-low power memory and logic devices as well as novel computing architectures. In this work, we employ synchrotron-based near-field infrared nano-spectroscopy to reveal the vibrational properties of ferroelastic (90 Ferroic domain walls are nano-objects that are considered functional elements in future devices. Here, the authors study phonons across ferroelastic domain walls by synchrotron-based near-field infrared nano-spectroscopy and relate these changes to the order parameter which helps to understand domain wall dynamics. Ferroics also have naturally occurring heterogeneities in the form of domains and domain walls18. This is especially true for Ca20 where the polarization arises from a trilinear coupling mechanism23 and abundant charged domain walls have been observed24. Atomic force and piezoforce imaging reveal the different orientations of directional order parameters and domain wall character, providing a physical playground for graph theory. Like other counterpart materials17, ferroelectric domain walls in Ca24. Theoretical modeling and electron diffraction experiments recently revealed that polarization rotates across the ferroelectric walls in a manner that is more Néel than Ising-like25. Ferroelastic walls in Ca20. Because the boundaries connect domains with different spontaneous strain states, mechanical compatibility conditions determine their orientation27. These ferroelastic walls are also 9020.Many unique properties in functional materials arise from spatially heterogeneous electronic and magnetic states. Examples include the formation of charge and spin stripes in some cuprates and nickelates and phase separation across the Mott transition of VO28. Second-harmonic generation spectroscopies also provide insight into local symmetries and chemical structure and, at the same time, are sensitive imaging tools8. X-ray photon correlation spectroscopy has uncovered ferroelectric domain wall dynamics in PbZr29. Despite a number of significant discoveries, almost nothing is known about domain wall phonons. Infrared spectroscopy, a powerful tool for probing phonons and their symmetries, has been unable to address the nano-scale heterogeneities of domain walls due to its poor spatial resolution. The long wavelengths of infrared light necessarily mean that, even for a diffraction-limited beam, traditional infrared techniques average over any micro- and nano-structured character of a target material, thereby reducing the sensitivity to these heterogeneities30. The development of near-field spectroscopic techniques has made real space imaging of nano-scale heterogeneity feasible, although most efforts are confined to the middle infrared due to the availability of suitable laser and broadband sources32. The extension into the far infrared using an accelerator-based source33 enables broadband measurement of the low-frequency response including phonons. Synchrotron-based near-field infrared spectroscopy34 thus has the potential to unlock the properties of defect states such as domain walls in complex oxides and chalcogenides. Near-field infrared spectroscopy is, of course, quite different than tip-enhanced Raman scattering in that ungerade (odd symmetry) phonon modes, which are crucial to the development of ferroelectricity and other functionalities, can be probed and analyzed.There is considerable interest in the dynamics of domain walls. Broadband scanning impedance microscopy of hexagonal ErMnO36. Loss mechanisms involving phonons are also key to controlling decoherence in domain wall-based computing architectures37.In order to uncover the behavior of fundamental excitations like phonons at domain walls and to explore the structural distortions that they represent, we performed far infrared synchrotron-based near-field nano-spectroscopy of the local phonon response at domain boundaries in Ca39. This structure  that transforms like the 24. For the chosen setting of the orthorhombic axes relative to the tetragonal axes, the twin domains are labeled by different settings of the space group symbol: 27. The wall symmetry is obtained by combining the symmetry elements of the two domains and those transforming one domain into the other. To go from twin A to twin B, all three structural order parameters rotate  and the other arm is a moving mirror. Synchrotron infrared light is focused and scattered off an AFM tip in close proximity to the sample surface. The scattered light is combined with the reference beam from the moving mirror on a silicon or KRS-5 beamsplitter and detected by a liquid helium-cooled Ge:Cu detector. Scanning the mirror of the reference arm creates an interferogram, which is Fourier-transformed to obtain both amplitude and phase information, which is related to the real and imaginary parts of the optical dielectric function of the material. The incident light is 49. To discriminate the near-field signal from the far-field background signal, the AFM is operated in non-contact (tapping) mode at a typical frequency of 250–300 kHz, and the corresponding detector signal is demodulated at twice the tapping frequency. Our measurements were performed with typical free oscillation tip amplitudes in the 80–100-nm range with a setpoint between 70% and 78% of the free-tapping amplitude, such that the engaged tapping amplitude values were in the 55–75-nm range. Routine approach curves with free oscillation tip amplitudes in the 80–100-nm range taken on gold indicate a near-field tip enhancement within Near-field infrared spectroscopy was performed using the set-ups at beamlines 5.4 and 2.4 at the Advanced Light Source, Lawrence Berkeley National LaboratoryOur near-field infrared measurements were carried out at room temperature over a frequency range between 330 and 800 cmWe also carried out traditional, far-field spectroscopy using a suite of Fourier transform and grating spectrometers covering the spectral range from 15 to 65,000 cm50 and the PBEsol functional, as implemented in VASP51. We used the  VASP pseudopotentials with electronic configurations 352. We performed group theoretic analysis with the aid of ISOTROPY53 and the Bilbao Crystallographic Server57 and visualized crystal structures using VESTA58.DFT calculations were performed using projector augmented wave pseudopotentialsSupplementary Information


BIRC6 lacking the UBC domain partially rescues the autophagosome-lysosome fusion defect in BIRC6-deficient cells, a role of BIRC6 in this event is independent of its E2 catalytic activity.The Inhibitor of Apoptosis (IAP) family member, Baculoviral IAP Repeat Containing 6 (BIRC6)/BRUCE is a ubiquitin conjugating E2 enzyme and a well-established anti-apoptosis regulator. However, its role in mammalian autophagy has not been shown. We identified BIRC6 as an important positive regulator of macroautophagy/autophagy by performing an siRNA screen targeting enzymes in the ubiquitin pathway. Compared to wild-type cells, BIRC6-deficient cells show accumulation of lipidated LC3B both at basal and starved conditions. Furthermore, BIRC6 deficiency blocks starvation-induced autophagic flux monitored by a tandem fluorescent autophagy sensor, mCherry-GFP-LC3B. Most strikingly, fusion of autophagosomes and lysosomes is blocked in BIRC6-deficient cells. BIRC6 colocalizes with the lysosomal protein LAMP2 in cells, and biochemically interacts with STX17 (syntaxin 17), which is a marker for completed autophagosomes. These data collectively suggest that BIRC6 bridges lysosomes and autophagosomes by interacting with these proteins. Because a deletion mutant of  Ubiquitination is a part of the regulatory mechanisms of autophagy, especially in selective types of autophagy such as mitophagy, xenophagy and aggrephagy. In selective autophagy, ubiquitinated cargos are brought to phagophores, the precursors to autophagosomes, via autophagy receptors such as SQSTM1/p62, bridging cargos and autophagic MAP1LC3/LC3 proteins on the phagophore. However, in general, only a little is understood about how enzymes in the ubiquitin pathway regulate the autophagic machinery.Atg7, Atg16l1, Birc6, Rb1cc1, Tsg101, Atg12, Sqstm1, Nedd8 and Atg9a), 5 of which are Atg (autophagy-related) genes implying that the screen was successful. Most importantly, we identified a previously unknown mammalian-autophagy regulator, BIRC6.To better understand a link between the ubiquitin system and autophagy, we aimed to identify enzymes in the ubiquitin pathway that regulate starvation-induced autophagy . To thisBaculoviral IAP Repeat Containing 6 (BIRC6)/BRUCE/Apollon is an inhibitor of apoptosis (IAP) family member and has a ubiquitin-conjugating E2 enzyme domain (UBC). BIRC6 is exceptionally large (more than 500 kDa) and is the only IAP family member to contain a UBC domain, whereas other members IAP1, IAP2 and XIAP are RING-type ubiquitin E3 ligases. Like other family members, BIRC6 inhibits apoptosis by degrading apoptosis regulators through the ubiquitin-proteasome system (UPS). In flies, Bruce has been implicated in autophagy and was suggested to be a negative regulator of autophagy. However, its role in mammalian autophagy has not been studied before.In BIRC6-deficient MEFs stably expressing mCherry-GFP-LC3B, we confirmed that loss of the starvation-induced GFP signal is impaired. Furthermore, both lipidated and non-lipidated forms of LC3B, GABARAP and GABARAPL1 are highly accumulated in BIRC6-deficient cells in comparison to wild-type cells. Starvation induces formation of LC3B-positive autophagosomes as well as mature autophagosomes positive for STX17 in both wild-type and BIRC6-deficient cells. These data collectively suggest that BIRC6 regulates autophagic flux without affecting formation and maturation of autophagosomes. We observed that starvation-induced autolysosome formation is drastically disturbed in BIRC6-deficient cells, and numbers of STX17-positive mature autophagosomes are highly increased at the basal condition in these cells, further supporting the idea that BIRC6 regulates autophagosome-lysosome fusion.Mechanistically, BIRC6 interacts with STX17 based on GST affinity-isolation assay, and colocalizes with the lysosomal protein LAMP2 as determined by confocal microscopy. Electron microscopy analysis suggests that transport of autophagosomes to lysosomes is not affected by BIRC6-deficiency. Based on these results, our working hypothesis is that BIRC6 bridges mature autophagosomes and lysosomes via STX17 and LAMP2, respectively, to promote their fusion .10.1080/Interestingly, BIRC6 selectively interacts with GABARAP and GABARAPL1 among the 6 LC3 family members  we analyzed. LC3 family proteins have homologous structures. It is not yet entirely clear if each member has unique and non-redundant functions in the regulation of autophagy; however, recent studies have suggested that GABARAPs have distinct functions from other members. For example, GABARAP proteins are more important than other LC3 proteins in autophagosome-lysosome fusion, and a pool of GABARAP resides on a centrosome region, which traffics from the Golgi to form autophagosomes. BIRC6 was also shown to localize with trans Golgi network markers in mammalian cells. Therefore, a BIRC6-GABARAP interaction might also be involved in autophagic trafficking processes.It would be an interesting question whether autophagy regulation by BIRC6 is totally independent from its role in the anti-apoptosis pathway. BIRC6 is associated with cancer in humans, and both cell death and autophagy are highly relevant in cancer biology. Thereby, understanding more detailed molecular mechanisms of how BIRC6 regulates cell death and autophagy may contribute to the development of therapies against cancer in the future.


Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate (PtdIns5P), which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.Tom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by  Cargo is further sorted and allocated into intralumenal vesicles in multivesicular bodies for later transportation and degradation in the lysosomal lumen. However, in some cases, ubiquitin-free receptors can be internalized in complex with their ligands to further propagate signaling from a unique population of endosomes known as signaling endosomes2. Ubiquitination is a signal for protein degradation by the proteasome machinery, but it can also lead to other nondegradative pathways3. Cargo sorting is mediated by ubiquitin-binding domains, which weakly bind to monomers or polymers of ubiquitin4. Ubiquitin-binding domains display higher selectivity and tighter binding to certain types of polyUb chains, where these polymers adopt either packed  or extended  conformations4.Cells dampen signaling through a broad range of mechanisms to control cellular processes, including ubiquitinated cell-surface receptor (cargo) internalization and delivery to the surface of early endosomes5. The canonical ESCRT-0 complex is composed of Hrs and STAM1/2 proteins6. Both Hrs and STAM1/2 bind cargo through their double-sided Ub-interacting motif (UIM) and VHS domains9. Anchoring of canonical ESCRT-0 to endosomal membranes is through PtdIns3P recognition via the FYVE domain of Hrs10. Functionally related proteins, including the endosomal target of Myb1 (Tom1), and its close relatives Tom1-like 1 and 2, Toll-interacting protein, and Endofin, have been proposed to work in parallel with ESCRT-0 as alternative ESCRT-0  early cargo transporters12. Like canonical ESCRT-0 proteins, alt-ESCRT-0 proteins have been reported to interact with ESCRT-I14, making it possible for them to interface with the remaining ESCRT apparatus. Tom1 is a multimodular protein composed of an N-terminal VHS domain, followed by a central GAT domain, and a long C-terminal region15. Both the VHS and GAT domains are involved in the recognition of cargo via their ubiquitin moieties18, whereas the C-terminal region has been reported to bind to the alt-ESCRT member, Endofin19.At the endosomal surface, cargo is transported through the endosomal sorting complex required for transport (ESCRT) apparatus. The ESCRT apparatus consists of four complexes named ESCRT-0, -I, -II, and –III, which sequentially transport cargo to intralumenal vesicles where cargo is loadedShigella flexneri, Salmonella thyphimurium, and Salmonella dublin, employ metabolic strategies to alter phosphatidylinositol 5-phosphate(PtdIns5P)levels to efficiently survive with the adverse environment in the host cell20. For example, S. flexneri invasion is thought to modify the overall organ oxygen consumption condition in the host21, consequently, making a more reducing cell environment. This condition would represent the major cause of the generation of hypoxia in the host21. S. flexneri invades host cells through internalization within a vacuole, then disrupting and escaping from it by delivering the virulent factor IpgD22, a lipid phosphatase that generates endosomal PtdIns5P, using cellular phosphatidylinositol 4,5-bisphosphate23. Accumulation of PtdIns5P has been reported to recruit Tom1 to signaling endosomes, thereby impairing both endosomal maturation and epidermal growth factor receptor (EGFR) degradation24. Endosomal recruitment of Tom1 relies on its N-terminal VHS domain, which preferentially binds PtdIns5P24. The aim of this study was to establish whether nonreducing and reducing environments and PtdIns5P binding influence the structure and, consequently, the function, of Tom1. The results identified a novel, irreversible, unfolded intermediate state in the Tom1 VHS domain under nonreducing conditions. Unlike phosphatidylcholine (PtdCho), the intermediate state of the protein was observed in the presence of PtdIns5P under both reducing and nonreducing conditions. However, PtdIns5P and related phospholipids, destabilized Tom1 VHS by reducing its thermostability and conformational compaction. Unlike ubiquitin, two PtdIns5P-binding sites, a high and a low affinity site with predominantly endothermic binding profiles, were identified in the Tom1 VHS domain with the acyl chain of the lipid playing an important role in the association. Results are discussed within the context of Tom1 recruitment to PtdIns5P-enriched endosomal compartments under bacterial infection conditions.Bacterial pathogens, including E.coli (Rosetta). The highest expression levels were achieved by induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubation of the cells for 4 h at 25 °C. Cells were lysed and the resulting cytoplasmic fraction was incubated with glutathione beads and the purified fusion protein cleaved with thrombin . Seventeen unique tryptic products of Tom1 VHS covered ~91% of the amino acid sequence , the locations of the cysteine residues in the VHS domain structure  and two others on helix 3 (C77 and C81) Fig. . The cryure Fig.  are ideaues Fig. . The proues Fig. . These r29. To better understand this behavior, we calculated its hydrophobicity using the Kyte and Doolittle hydrophobicity score30. Tom1 VHS displayed hydrophobicity in α-helices 2, 4, 5, 6, 7 and 8  , the far-UV CD spectrum of the protein slightly decreased in ellipticity with an estimated helical content of 62% and a 222/208 ratio of 0.957 ± 0.004 Fig. , incubatThe two tryptophan residues of the Tom1 VHS domain can serve as intrinsic probes for tracking interactions with protein ligands. Under nonreducing conditions, the protein exhibited a maximum tryptophan fluorescence emission at 343 nm. Gradual addition of PtdIns5P led to a reduction in fluorescence intensity of the Tom1 VHS domain without any evident change in the fluorescence spectrum maximum Fig.  but with8. STAM1 VHS recognizes the canonical hydrophobic patch at the surface of ubiquitin, represented by residues I44 and G47, which are completely buried at the complex interface8. Ubiquitin recognition is mediated by the α-helices α2 and α4 with W26 and L30 playing a major role in the interaction34. The equivalent residues in the Tom1 VHS domain are W30 and M34 P2], which is unable to form micelles. Binding of Tom1 VHS to InsP2 remained endothermic and best fit a single-site binding model, but the affinity for the headgroup was weak with a KD of 213 μM P2-binding sites has also been identified with atomic details in the yeast β-propeller PROPPIN protein, Hsv239. Further studies demonstrated that the PROPPIN family members Atg18, Atg21, and Hsv2 require both PtdIns3P/PtdInsP2-binding sites and an insertion loop, all located within a single domain, for membrane binding40. In some cases, such as for Atg18, phosphoinositides promote protein oligomerization and accommodate the oligomer perpendicularly to the membrane in a slightly tilted orientation41. Alternatively, two distinct phospholipid-binding sites are found in single protein domains such as PH, PX, and C2 domains43. This mechanism may serve as two independent points of membrane contacts, permitting coincidence detection. Thus, the low affinity PtdIns5P binding site in Tom1 VHS might represent a site for weak binding for the phosphoinositide or for another phospholipid. It is also possible that the second binding event constitutes a nonspecific PtdIns5P-binding site in the protein at excess of lipid. Nevertheless, additional molecular studies are needed to identify the PtdIns5P binding site/s in the Tom1 VHS domain. Our ITC analysis shows positive ∆H  values for the association of Tom1 VHS to PtdIns5P  experiments. Under nonreducing conditions, binding of water soluble PtdIns5P to Tom1 VHS was markedly endothermic with the resulting isotherms fitting a two-site binding model Fig. , in line μM Fig. , Table 35P Table . We specThe structural and thermodynamic results shown here provide new insights into phosphoinositide-binding domains and their behavior under nonreducing and reducing conditions. Specifically, thermal unfolding studies of the monomeric Tom1 VHS domain under nonreducing conditions show that the protein transitions through an intermediate state, which is absent under reducing conditions. Binding of Tom1 VHS to PtdIns5P leads to local conformational changes that reduce thermostability, interhelical contacts, and conformational compaction in the protein. Thus, conformation of the PtdIns5P-bound Tom1 VHS, found at a physiological temperature, may have a role in the progress of bacterial infection by facilitating yet-to-be-determined molecular interactions. In addition, the PtdIns5P-dependent conformational change of the VHS domain may explain why Tom1 is no longer engaged in ubiquitinated protein trafficking under bacterial infections. Our studies indicate that other phospholipids, including PtdIns3P, PtdIns, and PtdCho, also interact to and destabilize Tom1 VHS. Further, thermodynamic analysis suggests that Tom1 VHS displays two, both a high and a low, binding sites for PtdIns5P. Unlike ubiquitin binding, PtdIns5P binding events are markedly entropy-driven, noncooperative, and display distinct endothermic processes. Interestingly, the acyl chains of the phosphoinositide exhibit a relevant role for recognition, results that can explain the broad specificity of Tom1 VHS for phospholipids. Future studies are needed to understand the molecular mechanism by which Tom1 and its VHS domain associate to PtdIns5P-enriched membranes and to identify what regions in VHS undergo major conformational changes that trigger destabilization.45. A construct expressing human ubiquitin, cloned into the pET24d vector, was a gift from Dr. Julie Forman-Kay (University of Toronto). Ubiquitin was expressed and purified as described46.The human Tom1 VHS domain was cloned as previously reportedThe purified Tom1 VHS domain was loaded onto an FPLC system using a Superdex 75 column  equilibrated with 50 mM Tris-HCl (pH 7.3), and 500 mM NaCl in the absence and presence of 1 mM DTT. Protein standards for the Superdex 75 column were as follows: bovine serum albumin , ovalbumin , carbonic anhydrase , chymotrypsin A , cytochrome C , aprotinin , and vitamin B12 .3CN/H2O/CH3COOH  at 10 µM concentration. The protein solution was delivered with the aid of a syringe pump  at 0.5 µL/min to the ion source of an LTQ mass spectrometer . The sample was electrosprayed at 2.2 kV, and data acquisition was performed by averaging five scans per mass spectrum.Tom1 VHS was desalted with SPEC-PTC18 cartridges  and prepared for mass spectrometry (MS) analysis in a solution of CHDLS measurements were recorded at 25 °C using a Malvern Zetasizer Nano-ZS instrument. Tom1 VHS (0.8 mg/ml) was prepared in 20 mM HEPES (pH 7.4) and 150 mM KCl. Each measurement was collected for 120 s and three accumulated spectra were averaged with the protein previously equilibrated for 2 min. Data was analyzed using the Malvern Zetasizer software v. 7.11.47. All CD protein spectra were corrected for buffer background. Near-UV measurements were performed in 1 mm cells containing 200 μM Tom1 VHS in 5 mM sodium citrate (pH 7.3) and 50 mM KF with and without 1 mM DTT. A scan of buffer was subtracted from the corresponding averaged protein spectra. Temperature-dependent unfolding of Tom1 VHS was followed by changes of the CD signals at 222 nm as a function of increasing the temperature from 10 to 80 °C, with 1 °C steps and with a temperature gradient of 0.75 °C/min. All CD measurements were recorded in duplicate. To calculate the thermodynamic parameters, the derivatives of ellipticity as a function of temperature were calculated and fitted using CDpal48 following either two- or three-state denaturation models, as indicated. Thermal denaturation data were normalized between 0 and 1, with 1 denoting the unfolded protein value.Far-UV CD measurements were acquired on a Jasco J-815 spectropolarimeter with a temperature-controlled cell holder connected to a Peltier unit at the indicated temperatures. Tom1 VHS (20 μM) was prepared in 5 mM sodium citrate (pH 7.3) and 50 mM KF with and without 0.1–5 mM DTT. Three accumulated CD protein spectra, in the absence and presence of either dioctanoyl PtdIns5P (Echelon Biosciences) or dioctanoyl PtdCho (Avanti Lipids), were recorded in a 1-mm path length quartz cell using a bandwidth of 1-nm and a response time of 1 s at a scan speed of 50 nm/min. Secondary structure content of the Tom1 VHS domain, under different experimental conditions, was estimated using the CDSSTR algorithm available at the DICHROWEB serverIntrinsic tryptophan fluorescence measurements were collected using a Jasco J-815 spectropolarimeter. Emission spectra of the Tom1 VHS domain (0.25 μM) were recorded in 5 mM sodium citrate (pH 7.3) and 50 mM KF without or with 1.25 μM DTT, after excitation of the protein at 295 nm. Protein emission spectra, in the absence and presence of PtdIns5P, PtdIns3P, PtdIns, PtdCho, or ubiquitin, were collected between 310–410 nm using a 10-mm quartz cuvette at 25°C.Limited proteolysis with trypsin was carried out using 10 μg of purified Tom1 VHS in the absence or presence of either PtdIns5P, PtdIns3P, PtdIns, PtdCho, or ubiquitin (1:12 molar ratio). The mixtures were incubated for 5 min at room temperature. Then, trypsin  was added to the mixtures at amounts ranging from 0 to 5 μg for 30 min at 37 °C. The reactions were quenched by the addition of SDS-PAGE loading buffer and analyzed by 10% Tricine-SDS-PAGE and stained with Coomassie Brilliant Blue G-250 (VWR).2, or ubiquitin into buffer in a control measurement. The integrated heat data were fit with the one- P2) or two-binding (PtdIns5P) site models using the MicroCal PEAQ-ITC analysis software.ITC experiments were performed in 20 mM HEPES (pH 7.4) and 150 mM KCl. Proteins and lipids were dissolved in this buffer. Binding traces were analyzed with a MicroCal PEAQ  equilibrated at 25 °C. PtdIns5P (5 mM), PtdCho (5 mM), or ubiquitin (5 mM) were placed in the syringe, whereas the Tom1 VHS concentration in the sample cell for every titration was 50 μM. Titrations were carried out with 12-fold 3 μl injections. The first injection, which was 0.4 μl, was not considered for data analysis. All experiments were carried out at least in duplicate. The reported heats of binding were established by integrating the experimental peaks after adjusting the effect of heat dilution of either the lipids, InsPSupplementary Information


Tandem homologous domains in proteins are susceptible to misfolding through the formation of domain swaps, non-native conformations involving the exchange of equivalent structural elements between adjacent domains. Cutting-edge biophysical experiments have recently allowed the observation of tandem domain swapping events at the single molecule level. In addition, computer simulations have shed light into the molecular mechanisms of domain swap formation and serve as the basis for methods to systematically predict them. At present, the number of studies on tandem domain swaps is still small and limited to a few domain folds, but they offer important insights into the folding and evolution of multidomain proteins with applications in the field of protein design. Current Opinion in Structural Biology 2019, 58:97–104Biophysical and computational methodsThis review comes from a themed issue on Alan R Lowe and Laura S ItzhakiEdited by Issue and the EditorialFor a complete overview see the Available online 28th June 2019https://doi.org/10.1016/j.sbi.2019.05.012http://creativecommons.org/licenses/by/4.0/).0959-440X/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license  or across domains (inter-domain) •. TherefThe structure of each domain within a domain-swapped oligomer is essentially identical to the native fold and is characterised by the same backbone dihedral angles b. The onCloser analysis of tandem domain swaps also reveals fundamental similarities to so-called circular permutations c, cyclicWe are now in a position to better describe tandem domain swaps, which can be broken down into features of circular permutations and domain swaps. It is clear from the illustration in The first direct evidence of tandem domain misfolding came from Atomic Force Microscopy (AFM) experiments on tandem repeats of identical immunoglobulin-like I27 domains of titin •. In thii and j in a single titin domain as in the misfolded tandem dimer when the labels were conjugated instead to residues i and j in each of the adjacent domains of a titin dimer [More recently, single molecule Förster Resonance Energy Transfer (FRET) studies have been used to examine misfolding in tandem repeats of I27 ••,27••, Further time-resolved FRET experiments later revealed a complex mechanism for the formation of the domain-swapped misfolds ••. In adSo far, titin I27 domains have been the prototype for studies of tandem domain misfolding, but other recent experiments with these techniques suggest it is applicable to other domain folds, for example, in crystallins . AlthougMolecular dynamics simulations have long been used to explore the folding mechanisms of proteins, including the viability of circular permutations  and the Simulations have also proved useful in rationalizing why some tandem domains appear prone to domain-swapped misfolding, while others do not. In a more recent study, Tian and Best •• comparThe prediction of tandem domain swaps is fundamentally similar to the prediction of circular permutations and domain-swapped oligomers, which have been explored before. For circular permutations, prediction methods have centered on the identification of viable permutation positions in the domain sequence that would become the new termini ,22. SimiBuilding from the insights in the simulations by Tian and Best ••, we reed state . The inhThe direct estimation of the free energy allows the comparison of stabilities across different domains and is therefore more generally applicable than the estimation of relative probabilities. Furthermore, the distinction between the different steps (join and cut) allows the rationalization of predictions in terms of molecular determinants. For example, we can see in et al. [et al. [Thanks to the modularity of the alchemical free energy, the method is also applicable to evaluate circular permutations and domain-swapped oligomers with minor changes in the parameters . For exaet al. , hinge l [et al. •, and prDeterminants of misfolding in multidomain proteins are not yet fully understood. The studies presented here cover one type of misfolding event involving the formation of domain swaps in tandem homologous domains. Efforts from the experimental and computational side have established principles to understand the molecular mechanisms involved in tandem domain swapping, but the amount and diversity of available data are still limited. Nevertheless, the similarity of tandem domain swaps to other well studied domain folding variants, circular permutations and domain swapping in protein oligomerization, has in part permitted complex molecular simulations and their systematic prediction.One of the most evident gaps is the lack of experimentally solved structures of a tandem domain swap. There are sensible reasons for this absence, such as the low prevalence of tandem identical domain repeats in natural proteins, the selective reduction of repeats in crystallization constructs, or the transient nature and heterogeneity of tandem domain swap conformations. However, such a structure would be very helpful to set a precedent for future studies on tandem domain swaps. Given that the misfolded tandem domain-swapped state is in equilibrium with the native folded state, it will be necessary to design protein sequences that stabilise the misfolded conformation. One possibility might be the use of disulfide bridges, although this is not without complications. Alternatively, it should be possible to selectively introduce mutations which stabilise a specific swapped state in detriment of the native state. A possible avenue for rational design that has been successful in the past may be the use of models from coevolutionary sequence information . Other efolds which are likely to be prone to undergo tandem domain-swapping. Furthermore, TADOSS works under the assumption that the tandem domains are of identical sequence, but we know that the very large majority of tandem homologous domains are non-identical. For native multidomain proteins, in which the adjacent domains are homologous but not identical, it is known that the sequences of adjacent domains influence misfolding propensity [On the prediction side, the TADOSS method presented here only accounts for protein topology, that is, it predicts opensity •,27••. TProtein misfolding via tandem domain swapping can present major problems for the design and production of novel protein constructs. For instance, to increase avidity of interactions of designed protein biotherapeutics it would be convenient to simply duplicate the active part of protein. However, this may lead to increased protein misfolding if the sequence is not optimised to avoid tandem domain misfolds. Domain swapping has also been associated with the formation of amyloid fibrils involved in protein deposition diseases in the context of protein oligomerization , from whNational Institute of Diabetes and Digestive and Kidney Diseases (grant number ZIA DK075104-06) of the National Institutes of Health to P.T. and R.B.B. This work was funded by the European Molecular Biology Laboratory.This work was supported by the intramural research program of the Nothing declared.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


RNA-recognition motif (RRM) is an RNA-interacting protein domain that plays an important role in the processes of RNA metabolism such as the splicing, editing, export, degradation, and regulation of translation. Here, we present the RNA-recognition motif database (RRMdb), which affords rapid identification and annotation of RRM domains in a given protein sequence. The RRMdb database is compiled from ~57 000 collected representative RRM domain sequences, classified into 415 families. Whenever possible, the families are associated with the available literature and structural data. Moreover, the RRM families are organized into a network of sequence similarities that allows for the assessment of the evolutionary relationships between them. The RNA-recognition motif (RRM) domains are important players in the regulation of development , signaliThe evolution of RRM domains has been investigated in the context of various protein families. For example, structural studies of the heterogeneous nuclear ribonucleoprotein L (hnRNP L), a protein containing four RRM domains, showed that all four RRMs of hnRNP L contain a functional C-terminal extension , while both the second and third RRM domains additionally possess fifth β strand . Bioinfo10 function and values greater than 30 were capped to 30. MCL was started with the inflation parameter set to 2.0. To verify the robustness of the clustering procedure we performed two tests. First, we generated 10 000 artificial natural-like protein sequences using NullSeq  or the list of all families (`browse’ tab).Multi-RRM proteins are common regulators of alternative splicing  and the study of their origin and mutual relationships is important to understand how proteins increase their RNA-binding specificity and affinity. There are three possible evolutionary scenarios that could have led to the emergence of a multi-RRM protein (and to an orthologous family of multi-domain RRMs): (i) duplication of RRM domain(s) within a protein family, (ii) recombination or gene fusion resulting in domain shuffling between RRM-containing proteins and (iii) a combination of these two aforementioned events. The duplication might be supposed if RRM domains in a given protein family are more similar to each other than to any other RRM domains, whereas the recombination should be taken into account if RRM domains in a given protein family are more similar to other families than to each other. To distinguish between these two possibilities, it is necessary to assess the similarity between RRM domain families. Relying only on a pairwise sequence similarity value can be misleading; for example, two families that display low sequence similarity  may be, at the same time, their closest relatives that descended from a common ancestor. This problem can be addressed by applying an approach in which the evolutionary distance is approximated as the shortest path connecting two given RRM families in the network . The shoThe hnRNP L is a protein involved in many aspects of RNA metabolism and contains four RRM domains. The hnRNP L protein is a founding member of a family encompassing proteins with similar domain composition such as PTB, neural PTB and PTB 3 (Rod1). Despite the fact that the homology between the four RRMs of hnRNP L is barely detectable with BLAST, a recent study  has suggTypical SRSFs contain one or two RRM domains followed by a single low-complexity SR domain. In the work by Califice and colleagues  SRSF proRRMdb is proposed as a computational resource with which to study these and related events and to elucidate the complex evolution of RRM domains and RRM domain-containing proteins.


The phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in regulating cell growth and cell survival and is frequently deregulated in cancer cells. p85α regulates the p110α lipid kinase, and also stabilizes and stimulates PTEN, the lipid phosphatase that downregulates this pathway. In this report, we determined that the p85α BH domain binds several phosphorylated phosphoinositide lipids, an interaction that could help localize p85α to membranes rich in these lipids. We also identified key residues responsible for mediating PTEN – p85α complex formation. Based on these experimental results, a docking model for the PTEN – p85α BH domain complex was developed that is consistent with the known binding interactions for both PTEN and p85α. This model involves extensive side-chain and peptide backbone contacts between both the PASE and C2 domains of PTEN with the p85α BH domains. The p85α BH domain residues shown to be important for PTEN binding were p85α residues E212, Q221, K225, R228 and H234. We also verified experimentally the importance of PTEN-E91 in mediating the interaction with the p85α BH domain. These results shed new light on the mechanism of PTEN regulation by p85α. Thus, the balance of PI3K activity and PTEN activity determines Akt signaling output.The phosphatidylinositol 3-kinase (PI3K)/PTEN signaling pathway plays a central role in regulating cell cycle progression, cell growth, migration and survival –3. This 3,4,5P3) . PTEN is2 to PI3,4,5P3 to activate the PI3K pathway.The p85α protein serves as a regulatory protein for both p110α and PTEN. Monomeric p85α binds to p110α and stabilizes it, as well as constraining its PI3K activity , 8. Disi3 back to PI4,5P2 to inactivate PI3K pathway signaling [In response to upstream activation signals, PTEN is dephosphorylated within its regulatory domain, allowing it to form homodimers . The p85ignaling –14.3 levels resulting in PI3K pathway activation. Each of these instances results in enhanced downstream Akt signaling and the promotion of cell growth and cell survival [Aberrant activation of the PI3K/PTEN pathway can occur in cancer cells via activating mutations in p110α and/or p85α that relieve the inhibitory effects, yet maintain binding to promote p110α stability –19. AddiThe p85α protein contains 5 domains, an SH3 (Src Homology 3) domain capable of binding proline-rich sequences , a BH  , the phosphatase domain (PASE), the C2 domain, the regulatory domain (REG) and the PDZ binding region  Figure . Our pres Figure . C-termis Figure . All PTEAnalysis of the PTEN C2 domain suggests that it contains a protease-sensitive loop (residues 286–309) as suggested by the fact that it was deleted from the constructs used to obtain the crystal structure [Several PTEN C2 domain mutants were then generated and tested for p85α binding Figure . In eachOur initial characterization of the direct binding and regulation of PTEN by p85α showed that the BH domain of p85α was sufficient to mediate binding to PTEN . Using tE. coli as GST fusion proteins, and cleaved from GST  were selected for further characterization. To assess if the mutations altered the overall folding of the protein, circular dichroism (CD) spectroscopy was used to evaluate possible changes in protein secondary structure . The farPTEN lipid phosphatase activity is positively regulated by p85α binding , 13. To We previously reported solving the structure of the bovine p85α BH domain (105–319) , which wWe generated, crystalized and determined the structure of the p85α-BH-R228E protein (2.4 Å), containing a mutation in a potential sulfate binding site residue. In this crystal structure, one of the sulfate ions was clearly absent, and the remaining sulfate ion reoriented itself within the site Figure . No othe3 to PI4,5P2 [4 groups (6.8 Å) in inositol 1,3,4,5-tetrakisphosphate, raising the intriguing possibility that this surface may recognize phospholipid head groups. This is modeled in Figure The p85α protein is known to be a key regulatory protein of the PI3K/PTEN pathway; one of its mechanisms acting through binding to PTEN and facilitating the PTEN-mediated dephosphorylation of PI3,4,5P PI4,5P2 . We reco2, PI3,5P2, phosphatidic acid) >  > lysophosphatidic acid. Binding of p85α BH protein to the lipids immobilized on the blots was also found to be concentration dependent , p85α (78–319), or p85α (105–319), were used to probe lipid blots Figure . Bound pependent . The p853, whereas the binding to PI4,5P2 was about 2-fold weaker  for specific phospholipids, we carried out rotational anisotropy experiments using fluorescently labeled short-chain lipids Figure . In gener Figure . We alsor Figure . In contn Figure . For PI3y Figure . These ret al. suggest that p85α dimerization is most likely mediated by SH3 domain – proline-rich region interactions [Our extensive mutational analysis identified a number of key p85α BH domain residues important for binding of PTEN, including: D168, E212, E218, K224, K225, R228, S231, H234, L238, Q241 and K249 Figure . Similarractions , 22, as Taking all of this information into consideration, docking studies were performed using the ClusPro 2.0 server –34, inpu2 (PTEN – bovine p85α BH) and 1366 Å2 (PTEN – human p85α BH). The p85α BH domain residues that directly contact PTEN in the two docking models were not identical, however both models implicated E212, Q221, K225, R228, H234 and W237. Of these residues, four were part of the input data for the docking analysis, previously shown to be important for PTEN binding  . The R84E and Q221R mutations rendered these GST-PTEN proteins unstable, preventing us from assessing their p85α binding ability. The PTEN-E99R protein retained p85α binding ability, whereas the PTEN mutation E91R caused a significant reduction in p85α binding, providing additional support for our model Figure .Our PTEN deletion analysis suggested that the C2 domain of PTEN was involved in mediating binding to p85α. A previous report showed that the PTEN PASE domain may also be sufficient to mediate binding to p85α . Since tPairs of point mutations within the PTEN C2 domain demonstrated that mutation of K342E+K344E caused a marked reduction in the binding of full-length p85α. The model for PTEN – p85α BH domain docking does not position K342 and K344 at the interface of this contact surface. Thus, we speculate that these mutations may exert their effects either indirectly through allosteric changes in PTEN structure and/or via disrupting interactions between PTEN and other domains of p85α. PTEN mutations K342N  and K344R (prostate cancer)  have beeE. coli. The results obtained were similar though not identical, possibly due to the more complex environment within the cell lysates that allows for post-translation modifications and/or associations with cellular proteins, including dimerization with wild type p85α. The p85α BH domain mutations that showed reduced PTEN binding included D168R, E212R, K224E+K225E, R228E, H234D and Q241D. Some of these p85α mutations were also tested for PTEN activity modulation and, unexpectedly, several showed increased stimulation of PTEN lipid phosphatase activity . The reason for this result is not clear, but we speculate that these p85α mutants with reduced contacts between p85α and PTEN, may alter the relative orientation of the two proteins to impact PTEN lipid phosphatase reaction rates. It is also possible that the addition of p85α may help stabilize PTEN or could help solubilize the lipid substrate and thus, contribute to the increase in PTEN activity. The PTEN binding site is located on the opposite face of the p85α BH domain as compared to the GTPase/Rab5 binding site, consistent with previous reports that these sites are distinct [Our extensive mutagenesis of the p85α BH domain and subsequent PTEN binding analysis identified a number of residues that are important for PTEN binding. One analysis used mammalian cell lysates containing p85α mutants, whereas a second analysis used p85α mutant proteins purified from distinct .3. We used short-chain fluorescently labeled lipid head groups in an attempt to measure the affinity of lipid binding to the p85α BH domain. The affinity of these small lipids was extremely low, indicating that the short chain lipids do not interact with the p85α BH domain in solution, as they do when long chain lipids are used immobilized on a lipid blot, or possibly as part of a lipid bilayer. It is also possible that the interaction requires two adjacent lipids, as would be found on the surface of a membrane, to interact with two p85α BH domains within a p85α-p85α homodimer to produce sufficient avidity.We also identified a new lipid binding function for the p85α BH domain, with a preference for phosphorylated PI lipids such as PI3P and PI3,4,5P4 groups within inositol 1,3,4,5-tetrakisphosphate  also significantly reduced p85α binding. These results support the idea that these p85α and PTEN residues may help position the WPD loop of PTEN, containing catalytically important residues K92 and H93, and provide insight into the mechanism by which p85α binds and regulates PTEN.Our docking model also provides sufficient space for other PTEN and p85α domains not present in the modeled fragments, as well as interactions with other important binding partners. The N- and C-termini of both PTEN and p85α BH domain can be accommodated  . In addiA previous report carried out modeling using a similar docking strategy , but wit+/-)/p85α(+/-) mice compared to PTEN(+/-) mice [Pik3r1(-/-), the gene encoding p85α, not only showed decreased PTEN activity, elevated PI3,4,5P3 levels and increased Akt activation, they also developed liver tumors which progressed to metastatic cancer [Maintaining sufficient p85α to form p85α − PTEN complexes may have important implications for cancer development and progression. The incidence of intestinal polyps is increased two-fold in PTEN(/-) mice . Mice wic cancer . Ablatioc cancer . Thus, rIn summary, we have identified key residues within the p85α BH domain that define the PTEN binding site. We have used this experimental data to model the PTEN – p85α BH domain complex, and further tested this model by p85α mutagenesis and subsequent measurement of PTEN binding and lipid phosphatase activity regulation. The results of this study suggest that the p85α BH domain makes extensive contacts with PTEN and that critical p85α residues Q221, K224 and K225 play key catalytic roles in the stimulation of PTEN activity. The model sheds new light on the mechanism of PTEN regulation by p85α.Generation of the GST-PTEN plasmid has been described . The PTE2, 1 mM EDTA, 10 mM NaPPi, 100 mM NaF, + 10 µg/mL aprotinin, 10 µg/mL leupeptin and 1 mM AEBSF) and samples were centrifuged for 10 min at 4°C, 14000 x g to pellet cell debris. Lysates were precleared three times, each time using GST (100 μg) immobilized on glutathione Sepharose beads plus additional glutathione Sepharose beads (25 μl) for 1 hour at 4°C to remove non-specific binding proteins. Precleared supernants were analyzed by immunoblotting with anti-FLAG antibodies and were normalized for differences in FLAG-p85α protein expression by the addition of untransfected COS-1 cell lysates (prepared as above) to generate cell lysate samples containing similar levels of FLAG-p85α proteins. Lysates (5 μg in 200 μl) containing FLAG-p85α were used in each pull-down experiment with GST or GST-PTEN (5 μg each) immobilized on glutathione Sepharose beads. Samples were mixed for 30 minutes at 4°C and washed 5 times in 1 mL wash buffer . When using purified p85α proteins , they were precleared by incubating with GST (200 μg) immobilized on glutathione Sepharose beads overnight at 4°C to remove non-specific binding proteins. Each pull-down experiment used GST or GST-PTEN (5 μg each) immobilized on glutathione Sepharose beads incubated with precleared p85α protein ) for 30 minutes at room temperature. Beads were washed five times in 1 mL wash buffer. In both cases, bound p85α was detected after resolving samples by SDS-PAGE and transferring protein to nitrocellulose, by immunoblotting with anti-FLAG antibody  or anti- p85α antibody  as indicated. Secondary antibodies linked to Infrared-dyes  were used for detection and quantification using Odyssey software (v3.0). The results for all blots shown are representative of the results obtained from three independent experiments unless otherwise stated. Quantification of differences from wild type values were analyzed using a t-test with differences considered as statistically significant if P < 0.05.Pull-down assays with immobilized GST-PTEN were allowed to bind cell lysates containing FLAG-p85α or purified p85α protein (after cleavage from GST) in blocking as previously detailed . For exp6-PTEN (1 µM) with Di-C8-PI3,4,5P3 lipid  in the presence of wild type or mutant p85α (7.5 µM) in a final volume of 10 μl in phosphate release buffer . All incubations were performed at 37°C for 20 min and reactions were stopped with the addition of 100 mM N-ethylmaleimide . For detection, 20 μl of the reaction was combined with 80 μl BIOMOL Green (Biorad) in a 96-well plate and the color was allowed to develop for 20 min at room temperature. The absorbance was measured at 620 nm with a microplate reader. Each data point was assayed in duplicate, and all experiments were repeated five times. Values for buffer controls were subtracted from those for experimental samples and reported relative to PTEN activity in the presence of wild type p85α. Assay results are expressed as means ± standard errors. One-way analysis of variance (ANOVA) with post hoc Bonferroni tests were used for multiple comparisons with differences considered as statistically significant if P < 0.05. As a further control, we added bovine serum albumin to PTEN in this assay and found no change to PTEN activity, verifying that the activation of PTEN activity is specific to p85 (data not shown).A phosphate release assay was used to determine the effects of wild type and mutant p85α on PTEN activity as previously described . BrieflyCircular dichroism spectra were recorded for the p85α BH domain mutants that showed reduced binding to PTEN as compared to the wild type p85α protein to ensure that protein folding was retained, as described previously .myo-phosphatidylinositol (PI), D-myo-phosphatidylinositol 3-phosphate (PI3P), D-myo-phosphatidylinositol 4- phosphate (PI4P), D-myo-phosphatidylinositol 5-phosphate (PI5P), L-α-phosphatidylethanolamine (PE), L-α-phosphatidylcholine (PC), sphingosine 1-phosphate (S1P), D-myo-phosphatidylinositol 3,4-bisphosphate , D-myo-phosphatidylinositol 3,5-bisphosphate , D-myo-phosphatidylinositol 4,5-bisphosphate , D-myo-phosphatidylinositol 3,4,5-trisphosphate , L-α-phosphatidic acid (PA) and L- α -phosphatidylserine (PS). PIP strips were incubated in blocking buffer , in TBST ) for 1 hour at room temperature with agitation. The PIP strips were then incubated overnight at 4°C with 37 nM purified p85α protein fragment in blocking buffer, unless otherwise indicated. PIP strips treated with no protein were used as a control. PIP strips were washed in TBST  and incubated for 1 hour at 20°C with a p85α BH domain-specific antibody (0.7 μg/ml in blocking buffer) [Nitrocellulose-immobilized phospholipids  were lysophosphatidic acid (LPA), lysophosphocholine (LPC), D- buffer) . They weKd) by fitting to a rectangular hyperbola, so we used Sigma Plot 11.2 software to highlight the slope of each dataset as a straight line.Reaction mixtures (70 µl), contained Bodipy-FL labelled phosphatidylinositol lipids  in 50 mM Tris pH 7.0, 150 mM NaCl, 1 mM EDTA buffer. To this mixture, p85α BH domain wild type or mutant proteins were added ; samples were mixed and incubated for 15 seconds at 25°C following each addition. Rotational anisotropy was measured using a QuantaMaster QM-4 fluorometer  with a single emission channel for 10 seconds. Samples were excited with vertically polarized light at 503 nm, and both vertical and horizontal emissions were monitored at 513 nm (2.5-nm band pass). Since none of the binding curves reached saturation, it was not possible to determine dissociation constants  glycerol and the structure determined using X-ray diffraction data collected at the Canadian Light Source . Electron density was not present for residues 168–171 for both chains and residues 277–279 in Chain B. The atomic coordinates and diffraction intensities have now been deposited with the www Protein Data Bank and the corresponding deposition code is 6MRP.The purification, crystallization and structure determination for wild type and several mutants of the bovine p85α BH domain (105–319) has been described . The p85Coot, aided by the position of a vanadate ion and a tartrate ion observed in the active site of PTEN where the active site cysteine nucleophile (Cys124) forms a disulfide with Cys71 [The crystallographic coordinates for the human PTEN (PDB ID# 1D5R) and the human p85α BH domain (PDB ID# 1PBW) were obtained from the Protein Data Bank (PDB). Figures were generated using the PyMOL Molecular Graphics System . We modeled the structure of inositol 1,3,4,5-tetrakisphosphate (PDB ID# 1BWN)  in the nth Cys71 . The 3-pDocking analysis was performed using the ClusPro 2.0 server –34. The