PPXY Motif Research Articles

Smad7 Binds Differently to Individual and Tandem WW3 and WW4 Domains of WWP2 Ubiquitin Ligase Isoforms.

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein–protein interaction WW domains. The WW4 domain-containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial–mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-ΔHECT) comprising WW3–WW4 tandem domains and a truncated HECT domain that can inhibit TGFβ signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis.

Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth

ABSTRACTUVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications.Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; BECN1: beclin 1; CHX: cycloheximide; CSNK1A1/CK1α: casein kinase 1 alpha 1; CQ: chloroquine; DUB: deubiquitinase; EBSS: Earle’s balanced salt solution; EGF: epidermal growth factor; GFP: green fluorescent protein; GST: glutathione S-transferase; HBSS: Hanks balanced salts solution; HCC: hepatocellular carcinoma; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryo fibroblasts; mRFP: monomeric red fluorescent protein; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTMs: post-translational modifications; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; Ub-AMC: ubiquitin-7-amido-4-methylcoumarin: a fluorogenic substrate; UVRAG: UV radiation resistance associated; ZRANB1/TRABID: zinc finger RANBP2-type containing 1

Delineating WWOX Protein Interactome by Tandem Affinity Purification-Mass Spectrometry: Identification of Top Interactors and Key Metabolic Pathways Involved.

It has become clear from multiple studies that WWOX (WW domain-containing oxidoreductase) operates as a “non-classical” tumor suppressor of significant relevance in cancer progression. Additionally, WWOX has been recognized for its role in a much wider array of human pathologies including metabolic conditions and central nervous system related syndromes. A myriad of putative functional roles has been attributed to WWOX mostly through the identification of various binding proteins. However, the reality is that much remains to be learned on the key relevant functions of WWOX in the normal cell. Here we employed a Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach in order to better define direct WWOX protein interactors and by extension interaction with multiprotein complexes under physiological conditions on a proteomic scale. This work led to the identification of both well-known, but more importantly novel high confidence WWOX interactors, suggesting the involvement of WWOX in specific biological and molecular processes while delineating a comprehensive portrait of WWOX protein interactome. Of particular relevance is WWOX interaction with key proteins from the endoplasmic reticulum (ER), Golgi, late endosomes, protein transport, and lysosomes networks such as SEC23IP, SCAMP3, and VOPP1. These binding partners harbor specific PPXY motifs which directly interact with the amino-terminal WW1 domain of WWOX. Pathway analysis of WWOX interactors identified a significant enrichment of metabolic pathways associated with proteins, carbohydrates, and lipids breakdown. Thus, suggesting that WWOX likely plays relevant roles in glycolysis, fatty acid degradation and other pathways that converge primarily in Acetyl-CoA generation, a fundamental molecule not only as the entry point to the tricarboxylic acid (TCA) cycle for energy production, but also as the key building block for de novo synthesis of lipids and amino acids. Our results provide a significant lead on subsets of protein partners and enzymatic complexes with which full-length WWOX protein interacts with in order to carry out its metabolic and other biological functions while also becoming a valuable resource for further mechanistic studies.

Cancerous Protein Network That Inhibits the Tumor Suppressor Function of WW Domain-Containing Oxidoreductase (WWOX) by Aberrantly Expressed Molecules.

Recent findings indicate that the WW domain-containing oxidoreductase (WWOX) is a tumor suppressor protein that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. WWOX protein mediates multiple signaling networks that suppress carcinogenesis through binding of its first WW domain to various cancer-associated proteins, i.e., p73, AP-2γ, and others. Although the tumor suppressor property of WWOX is inarguable, WWOX is not inactivated in the manner characteristic of the canonical Knudson hypothesis. Impairment of both alleles of WWOX is thought to be a rare event, only occurring in a few cancer cell lines. How is the tumor suppressor function of WWOX impaired in cancer cells? Recent advances highlight that a small transmembrane protein possessing a PPxY motif, called TMEM207, and its relatives are aberrantly expressed in various cancer cells and hinder the tumor suppressor function of WWOX through inhibiting its WW domain. Here, we review the recent findings related to the pathobiological properties of TMEM207 and its relatives based on clinicopathological and experimental pathological studies.

WBP2 shares a common location in mouse spermatozoa with WBP2NL/PAWP and like its descendent is a candidate mouse oocyte-activating factor.

The sperm-borne oocyte-activating factor (SOAF) resides in the sperm perinuclear theca (PT). A consensus has been reached that SOAF most likely resides in the postacrosomal sheath (PAS), which is the first region of the PT to solubilize upon sperm–oocyte fusion. There are two SOAF candidates under consideration: PLCZ1 and WBP2NL. A mouse gene germline ablation of the latter showed that mice remain fertile with no observable phenotype despite the fact that a competitive inhibitor of WBP2NL, derived from its PPXY motif, blocks oocyte activation when coinjected with WBP2NL or spermatozoa. This suggested that the ortholog of WBP2NL, WBP2, containing the same domain and motifs associated with WBP2NL function, might compensate for its deficiency in oocyte activation. Our objectives were to examine whether WBP2 meets the developmental criteria established for SOAF and whether it has oocyte-activating potential. Immunoblotting detected WBP2 in mice testis and sperm and immunofluorescence localized WBP2 to the PAS and perforatorium of the PT. Immunohistochemistry of the testes revealed that WBP2 reactivity was highest in round spermatids and immunofluorescence detected WBP2 in the cytoplasmic lobe of elongating spermatids and colocalized it with the microtubular manchette during PT assembly. Microinjection of the recombinant forms of WBP2 and WBP2NL into metaphase II mouse oocytes resulted in comparable rates of oocyte activation. This study shows that WBP2 shares a similar testicular developmental pattern and location with WBP2NL and a shared ability to activate the oocyte, supporting its consideration as a mouse SOAF component that can compensate for a WBP2NL.

Ubiquitin Regulation of Trk Receptor Trafficking and Degradation.

The regulation of Trk receptors is critical for orchestrating multiple signalling pathways required for developing and maintaining neuronal networks. Activation of Trk receptors results in signalling, internalisation and subsequent degradation of the protein. Although ubiquitination of TrkA by Nedd4-2 has been identified as an important degradation pathway, much less is known about the pathways regulating the degradation of TrkB and TrkC. Critical to the interaction between TrkA and Nedd4-2 is a PPxY motif present within TrkA but absent in TrkB and TrkC. Given the absence of this interaction motif, it remains to be determined how TrkB and TrkC are ubiquitinated. Here we report that the adaptor protein Ndfip1 can interact with all three Trk receptors and show for TrkB the recruitment of Nedd4-2 through PPxY motifs present in Ndfip1. Ndfip1 mediates the ubiquitination of TrkB, resulting in receptor trafficking predominantly on Rab7 containing late endosomes, highlighting a pathway for TrkB degradation at the lysosome. In vitro, overexpression of Ndfip1 increased TrkB ubiquitination and decreased viability of BDNF-dependent primary neurons. In vivo, conditional genetic deletion of Ndfip1 increased TrkB in the brain and resulted in enlargement of the granular cell layer of the dentate gyrus.

Biophysical studies and modelling indicate the binding preference of TAZ WW domain for LATS1 PPxY motif

The Hippo tumor suppressor pathway is an important regulator of cell proliferation and apoptosis, and signal transduction occurs through phosphorylation of the effector protein TAZ by the serine/threonine kinase LATS1/2. Here, we report the biophysical and computational studies to characterize the interaction between TAZ and LATS1/2 through WW domain-PPxY motif binding. We show that the TAZ WW domain exhibits a binding preference for the second of the two PPxY motifs of LATS1 in vitro. We modelled the structure of the domain in complex with LATS1 PPxY2 peptide and, through molecular dynamics simulations, show that WW domain-PPxY2 complex is stable with some flexibility in the peptide region. Next, we predict and verify that L143 and T150 of the WW domain are important for TAZ binding with the PPxY2 peptide using mutational and isothermal titration calorimetric studies. Furthermore, we suggest that the electrostatic potential of charged residues within the binding pocket may influence the ligand affinity among otherwise highly similar WW domains.

The pUL56 of pseudorabies virus variant induces downregulation of swine leukocyte antigen class I molecules through the lysosome pathway

Pseudorabies virus (PRV) is the causative agent of pseudorabies (PR) which causes large economic losses for Chinese swine industry since breaking out in late 2011. As a member of herpesviruses, PRV is able to escape the host immune elimination and establish latency, resulting in persistent infection. Here, we report that a currently prevalent Chinese PRV variant down-regulated swine leukocyte antigen class I (SLA-I) molecules on the surface of PK-15 cells and targeted them for degradation through lysosome pathway. Viral pUL56 protein, independent of other viral proteins, was associated with this function by inducing degradation of cellular SLA-I heavy chain (HC) in a manner that was dependent on the lysosome machinery. In addition, pUL56 interacted with SLA-I HC and increased its ubiquitination. Further studies demonstrated that the late domains (PPXY motifs) of pUL56 were required for the ubiquitination and degradation of SLA-I HC by pUL56. Together, our findings reveal the mechanisms by which PRV interferes with cytotoxic T lymphocyte (CTL) responses and provide novel insights into the roles of PRV pUL56.

Activation and auto‐destruction of the ubiquitin ligase Itch

Ubiquitin ligases of the Nedd4 family are maintained inactive by intramolecular interactions (1). We reported that, like Nedd4, the closely related ubiquitin ligase Itch strongly binds to the endosomal protein LITAF through its WW domains, that recognize two PPXY motifs in the N‐terminal half of LITAF. Unlike Nedd4, Itch binding to LITAF brings the ligase to the lysosomal compartments. We thus postulated that LITAF overexpression might bring Itch to the lysosome, where it would access different substrates and target them for degradation (2). Concomitantly, LITAF binding to Itch may increase Itch ligase activity, as other PPXY motif proteins such as NDFIP and LRAD3 (3,4). Here, we set up to determine if Itch ubiquitin ligase activity is indeed up regulated by LITAF binding, and if Itch most common substrate degradation is increased in response to LITAF binding to Itch. We find that Itch, WWP1, WWP2 and Nedd4 all bind to LITAF, and that the four ligases autoubiquitylation is greatly increased upon LITAF binding, albeit to different levels. Both LITAF overexpression and addition of recombinant LITAF to crude cell lisates lead to rapid degradation of Itch, and, to a lesser extent, Nedd4. Itch substrates cJun and Endophilin degradation was also increased. In vitro ubiquitylation assays show that Itch autoubiquitylation is enhanced in the presence of LITAF, but substrate ubiquitylation was not significantly increased. In conclusion, LITAF binding strongly activate Itch autoubiquitylation, as well as that of other ligases of the Nedd4 family. The consequence of this activation is Itch proteasomal degradation, with little effect on its substrates ubiquitylation. LITAF is thus likely to be a negative regulator of Itch in cells where it is strongly expressed.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proline Restricts Loop I Conformation of the High Affinity WW Domain from Human Nedd4-1 to a Ligand Binding-Competent Type I β-Turn.

Sequence alignment of the four WW domains from human Nedd4-1 (neuronal precursor cell expressed developmentally down-regulated gene 4-1) reveals that the highest sequence diversity exists in loop I. Three residues in this type I β-turn interact with the PPxY motif of the human epithelial Na+ channel (hENaC) subunits, indicating that peptide affinity is defined by the loop I sequence. The third WW domain (WW3*) has the highest ligand affinity and unlike the other three hNedd4-1 WW domains or other WW domains studied contains the highly statistically preferred proline at the ( i + 1) position found in β-turns. In this report, molecular dynamics simulations and experimental data were combined to characterize loop I stability and dynamics. Exchange of the proline to the equivalent residue in WW4 (Thr) results in the presence of a predominantly open seven residue Ω loop rather than the type I β-turn conformation for the wild-type apo-WW3*. In the presence of the ligand, the structure of the mutated loop I is locked into a type I β-turn. Thus, proline in loop I ensures a stable peptide binding-competent β-turn conformation, indicating that amino acid sequence modulates local flexibility to tune binding preferences and stability of dynamic interaction motifs.

Herpesviruses possess conserved proteins for interaction with Nedd4 family ubiquitin E3 ligases

Nedd4 is a family of ubiquitin E3 ligases that regulate numerous cellular processes. In this report, we showed that alpha- and beta-herpesviruses have membrane proteins that regulate the function of the Nedd4 family members. Although the homology search score was quite low, UL56 of herpes simplex virus type 1 and 2, ORF0 of varicella-zoster virus, UL42 of human cytomegalovirus, and U24 of human herpesvirus 6A, 6B, and 7 all possess at least one PPxY (PY) motif in their cytoplasmic domain, and are able to bind with Itch, a member of the Nedd4 family. These viral proteins altered the localization of Itch and decreased Itch expression in co-expressing cells. In addition, these viral proteins reduced the production of retrovirus vectors through the regulation of the Nedd4 family of proteins. U24, but not the other proteins, effectively reduced CD3ε expression on the T cell surface. These viral molecules are thought to contribute to the specific function of each virus through the regulation of Nedd4 family activity.

Ehrlichia chaffeensis TRP32 Nucleomodulin Function and Localization Is Regulated by NEDD4L-Mediated Ubiquitination.

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate various host cell processes. In a previous study, we reported that the E. chaffeensis nucleomodulin TRP32 regulates transcription of host genes in several biologically relevant categories, including cell differentiation and proliferation. In this study, we investigate the effect of ubiquitination on TRP32 function and localization within the host cell. TRP32 is both mono- and polyubiquitinated on multiple lysine residues during infection and when ectopically expressed. Despite lacking a canonical PPxY motif, TRP32 interacted with, and was modified by the human HECT E3 ubiquitin (Ub) ligase NEDD4L. TRP32 ubiquitination was not by K48-linked polyUb chains, nor was it degraded by the proteasome; however, TRP32 was modified by K63-linked polyUb chains detected both in the cytosol and nucleus. HECT ligase inhibitor, heclin, altered the subnuclear localization of ectopically expressed TRP32 from a diffuse nuclear pattern to a lacy, punctate pattern with TRP32 distributed around the periphery of the nucleus and nucleoli. When a TRP32 lysine null (K-null) mutant was ectopically expressed, it exhibited a similar phenotype as single lysine mutants (K63R, K93R, and K123R). However, the K-null mutant showed increased amounts of cytoplasmic TRP32 compared to single lysine mutants or heclin-treated cells ectopically expressing TRP32. These alterations in localization corresponded to changes in TRP32 transcriptional repressor function with heclin-treated and single lysine mutants unable to repress transcription of a TRP32 target genes in a luciferase assay.

Differential Binding Affinities and Allosteric Conformational Changes Underlie Interactions of Yorkie and a Multivalent PPxY Partner.

Tondu domain-containing growth inhibitor (Tgi) is one of a growing number of multivalent PPxY proteins that regulate cell growth via interactions with the tandem WW domains of the transcription coactivator protein, Yorkie (Yki). These proteins are attractive candidates for targeted drug design, but the substantial amount of disorder predicted from their primary sequences makes structural studies that are foundational to drug design challenging. We have successfully overexpressed full length recombinant Tgi and Yki, experimentally confirmed that intrinsic structural disorder is common to both proteins, and assessed binding of the Yki WW domains to the three Tgi PPxY motifs using nuclear magnetic resonance and isothermal titration calorimetry. We find that the tandem WW domains positively cooperate to engage all three PPxY sites with a broad range of affinities. The first PPxY motif that is quite distant from the other two serves as the "binding initiation" site and is essential for high-affinity interactions. Importantly, by monitoring binding to the full length or larger protein domains, we obtain more physiologically relevant affinity information and identify "long-range" residues that could be targeted to fine-tune binding. This expansion of protein functionality through modulation of residues outside the recognition sequences offers potential alternative targets for drug design.

Biophysical studies and NMR structure of YAP2 WW domain - LATS1 PPxY motif complexes reveal the basis of their interaction.

YES-associated protein (YAP) is a major effector protein of the Hippo tumor suppressor pathway, and is phosphorylated by the serine/threonine kinase LATS. Their binding is mediated by the interaction between WW domains of YAP and PPxY motifs of LATS. Their isoforms, YAP2 and LATS1 contain two WW domains and two PPxY motifs respectively. Here, we report the study of the interaction of these domains both in vitro and in human cell lines, to better understand the mechanism of their binding. We show that there is a reciprocal binding preference of YAP2-WW1 with LATS1-PPxY2, and YAP2-WW2 with LATS1-PPxY1. We solved the NMR structures of these complexes and identified several conserved residues that play a critical role in binding. We further created a YAP2 mutant by swapping the WW domains, and found that YAP2 phosphorylation at S127 by LATS1 is not affected by the spatial configuration of its WW domains. This is likely because the region between the PPxY motifs of LATS1 is unstructured, even upon binding with its partner. Based on our observations, we propose possible models for the interaction between YAP2 and LATS1.

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress.IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.

Smurf Downregulates Echinoid in the Amnioserosa To Regulate Drosophila Dorsal Closure.

Drosophila dorsal closure is a morphogenetic movement that involves flanking epidermal cells, assembling actomyosin cables, and migrating dorsally over the underlying amnioserosa to seal at the dorsal midline. Echinoid (Ed)-a cell adhesion molecule of adherens junctions (AJs)-participates in several developmental processes. The disappearance of Ed from the amnioserosa is required to define the epidermal leading edge for actomyosin cable assembly and coordinated cell migration. However, the mechanism by which Ed is cleared from amnioserosa is unknown. Here, we show that Ed is cleared in amnioserosa by both transcriptional and post-translational mechanisms. First, Ed mRNA transcription was repressed in amnioserosa prior to the onset of dorsal closure. Second, the ubiquitin ligase Smurf downregulated pretranslated Ed by binding to the PPXY motif of Ed. During dorsal closure, Smurf colocalized with Ed at AJs, and Smurf overexpression prematurely degraded Ed in the amnioserosa. Conversely, Ed persisted in the amnioserosa of Smurf mutant embryos, which, in turn, affected actomyosin cable formation. Together, our results demonstrate that transcriptional repression of Ed followed by Smurf-mediated downregulation of pretranslated Ed in amnioserosa regulates the establishment of a taut leading edge during dorsal closure.

Molecular basis of interactions between SH3 domain-containing proteins and the proline-rich region of the ubiquitin ligase Itch

The ligase Itch plays major roles in signaling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding are critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPXY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pulldown assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with endophilins, amphyphisins, and pacsins but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modeling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with β-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR toward its most common SH3 domain-containing partners and demonstrate that the PRR region is sufficient for binding.

Interactions of U24 from Roseolovirus with WW domains: canonical vs noncanonical

U24 is a C-terminal membrane-anchored protein found in both human herpes virus type 6 and 7 (HHV-6 and HHV-7), with an N-terminal segment that is rich in prolines (PPxY motif in both HHV-6A and 7; PxxP motif in HHV-6A). Previous work has shown that U24 interacts strongly with Nedd4 WW domains, in particular, hNedd4L-WW3*. It was also shown that this interaction depends strongly on the nature of the amino acids that are upstream from the PY motif in U24. In this contribution, data was obtained from pull-downs, isothermal titration calorimetry, and NMR to further determine what modulates U24:WW domain interactions. Specifically, 3 non-canonical WW domains from human Smad ubiquitination regulatory factor (Smurf), namely hSmurf2-WW2, hSmurf2-WW3, and a tandem construct hSmurf2-WW2 + 3, were studied. Overall, the interactions between U24 and these Smurf WW domains were found to be weaker than those in U24:Nedd4 WW domain pairs, suggesting that U24 function is tightly linked to specific E3 ubiqitin ligases.

Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid.

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.