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Low-grade Oncocytic Tumor (LOT) of the kidney is a recently described entity with poorly understood pathogenesis. Using next-generation sequencing (NGS) and complementary approaches, we provide insight into its biology. We describe 22 LOT corresponding to 7 patients presenting with a median age of 75 years (range 63–86 years) and male to female ratio 2:5. All 22 tumors demonstrated prototypical microscopic features. Tumors were well circumscribed and solid. They were composed of sheets of tumor cells in compact nests. Tumor cells had eosinophilic cytoplasm, round to oval nuclei (without nuclear membrane irregularities), focal subtle perinuclear halos and occasional binucleation. Sharply delineated edematous stromal islands were often observed. Tumor cells were positive for PAX8, negative for CD117 and exhibited diffuse and strong cytokeratin-7 expression. Six patients presented with pT1 tumors and at a median follow-up of 29 months, 4 patients were alive without recurrence (3 patients had died from unrelated causes). All tumors were originally classified as chromophobe renal cell carcinoma, eosinophilic variant (chRCC-eo). While none of the patients presented with known syndromic features, one patient with multiple bilateral LOTs was subsequently found to have a likely pathogenic germline TSC1 mutation. Somatic, likely activating, mutations in MTOR and RHEB were identified in all other evaluable LOTs. As assessed by phospho-S6 and phospho-4E-BP1, mTOR complex 1 (mTORC1) was activated across all cases but to different extent. MTOR mutant LOT typically exhibited lower levels of mTORC1 activation, possibly related to mTORC1 dimerization and the preservation of a wild-type MTOR copy (retained chromosome 1). Supporting its distinction from related entities, gene expression analyses showed that LOT clustered separately from classic chRCC, chRCC-eo and RO. In summary, converging mTORC1 pathway mutations, mTORC1 complex activation and a distinctive gene expression signature along with characteristic phenotypic features support LOT designation as a distinct entity with both syndromic and non-syndromic cases associated with an indolent course.

Table S1 - HER2 mutations Fig S1 - HER2 co-occur Fig S2 - Long Term Fulv. Fig S3 - L755S CI index Fig S4 - L755S RTprofile Fig S5 - Prot Align Fig S6 - Drug Assays Fig S7 - Quant of Drug Fig S8-11 - immunoblot Fig S12 - L755S mouse Fig S13 - S6 IHC - mouse

Purpose:We examined the role of ERBB2-activating mutations in endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer.Experimental Design:ERBB2 mutation frequency was determined from large genomic databases. Isogenic knock-in ERBB2 mutations in ER+ MCF7 cells and xenografts were used to investigate estrogen-independent growth. Structural analysis was used to determine the molecular interaction of HERL755S with HER3. Small molecules and siRNAs were used to inhibit PI3Kα, TORC1, and HER3.Results:Genomic data revealed a higher rate of ERBB2 mutations in metastatic versus primary ER+ tumors. MCF7 cells with isogenically incorporated ERBB2 kinase domain mutations exhibited resistance to estrogen deprivation and to fulvestrant both in vitro and in vivo, despite maintaining inhibition of ERα transcriptional activity. Addition of the irreversible HER2 tyrosine kinase inhibitor neratinib restored sensitivity to fulvestrant. HER2-mutant MCF7 cells expressed higher levels of p-HER3, p-AKT, and p-S6 than cells with wild-type HER2. Structural analysis of the HER2L755S variant implicated a more flexible active state, potentially allowing for enhanced dimerization with HER3. Treatment with a PI3Kα inhibitor, a TORC1 inhibitor or HER3 siRNA, but not a MEK inhibitor, restored sensitivity to fulvestrant and to estrogen deprivation. Inhibition of mutant HER2 or TORC1, when combined with fulvestrant, equipotently inhibited growth of MCF7/ERBB2V777L xenografts, suggesting a role for TORC1 in antiestrogen resistance induced by ERBB2 mutations.Conclusions:ERBB2 mutations hyperactivate the HER3/PI3K/AKT/mTOR axis, leading to antiestrogen resistance in ER+ breast cancer. Dual blockade of the HER2 and ER pathways is required for the treatment of ER+/HER2 mutant breast cancers.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

An evolutionary-based definition and classification of target evaluation units (EUs) is presented for the 14th round of the critical assessment of structure prediction (CASP14). CASP14 targets included 84 experimental models submitted by various structural groups (designated T1024-T1101). Targets were split into EUs based on the domain organization of available templates and performance of server groups. Several targets required splitting (19 out of 25 multidomain targets) due in part to observed conformation changes. All in all, 96 CASP14 EUs were defined and assigned to tertiary structure assessment categories (Topology-based FM or High Accuracy-based TBM-easy and TBM-hard) considering their evolutionary relationship to existing ECOD fold space: 24 family level, 50 distant homologs (H-group), 12 analogs (X-group), and 10 new folds. Principal component analysis and heatmap visualization of sequence and structure similarity to known templates as well as performance of servers highlighted trends in CASP14 target difficulty. The assigned evolutionary levels (i.e., H-groups) and assessment classes (i.e., FM) displayed overlapping clusters of EUs. Many viral targets diverged considerably from their template homologs and thus were more difficult for prediction than other homology-related targets. On the other hand, some targets did not have sequence-identifiable templates, but were predicted better than expected due to relatively simple arrangements of secondary structural elements. An apparent improvement in overall server performance in CASP14 further complicated traditional classification, which ultimately assigned EUs into high-accuracy modeling (27 TBM-easy and 31 TBM-hard), topology (23 FM), or both (15 FM/TBM).

Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase deamidating activity of VopC, a type three secretion system (T3SS) translocated effector, drives V. parahaemolyticus invasion. The intracellular pathogen uropathogenic Escherichia coli (UPEC) invades host cells by secreting a VopC homolog, the secreted toxin cytotoxic necrotizing factor one (CNF1). Because of the homology between VopC and CNF1, we hypothesized topical application of CNF1 during V. parahaemolyticus infection could supplement VopC activity. Here, we demonstrate that CNF1 improves the efficiency of V. parahaemolyticus invasion, a bottleneck in V. parahaemolyticus infection, across a range of doses. CNF1 increases V. parahaemolyticus invasion independent of both VopC and the T3SS altogether, but leaves a disproportionate fraction of intracellular bacteria unable to escape the endosome and complete their infection cycle. This phenomenon holds true in the presence or absence of VopC, but is particularly pronounced in the absence of a T3SS. The native VopC, by contrast, promotes a far less efficient invasion, but permits the majority of internalized bacteria to escape the endosome and complete their infection cycle. These studies highlight the significance of enzymatic specificity during infection, as virulence factors (VopC and CNF1 in this instance) with similarities in function (bacterial uptake), catalytic activity (deamidation), and substrates (Rho GTPases) are not sufficiently interchangeable for mediating a successful invasion for neighboring bacterial pathogens.IMPORTANCEMany species of intracellular bacterial pathogens target host small Rho-GTPases to initiate invasion, including the human pathogens Vibrio parahaemolyticus and uropathogenic Eschericia coli (UPEC). The type three secretion system (T3SS) effector VopC of V. parahaemolyticus promotes invasion through the deamidation of Rac1 and CDC42 in the host, whereas the secreted toxin cytotoxic necrotizing factor one (CNF1) drives UPEC’s internalization through the deamidation of Rac1, CDC42, and RhoA. Despite these similarities in the catalytic activity of CNF1 and VopC, we observed the two enzymes were not interchangeable. Although CNF1 increased V. parahaemolyticus endosomal invasion, most intracellular V. parahaemolyticus aborted their infection cycle and remained trapped in endosomes. Our findings illuminate how the precise biochemical fine-tuning of T3SS effectors is essential for efficacious pathogenesis. They moreover pave the way for future investigations into the biochemical mechanisms underpinning V. parahaemolyticus endosomal escape, and more broadly, the regulation of successful pathogenesis.

This report describes the tertiary structure prediction assessment of difficult modeling targets in the 14(th) round of the Critical Assessment of Structure Prediction (CASP14). We implemented an official ranking scheme that used the same scores as the previous CASP topology-based assessment, but combined these scores with one that emphasized physically realistic models. The top performing AlphaFold2 group outperformed the rest of the prediction community on all but two of the difficult targets considered in this assessment. They provided high quality models for most of the targets (86% over GDT_TS 70), including larger targets above 150 residues, and they correctly predicted the topology of almost all the rest. AlphaFold2 performance was followed by two manual Baker methods, a Feig method that refined Zhang-server models, two notable automated Zhang server methods (QUARK and Zhang-server), and a Zhang manual group. Despite the remarkable progress in protein structure prediction of difficult targets, both the prediction community and AlphaFold2, to a lesser extent, faced challenges with flexible regions and obligate oligomeric assemblies. The official ranking of top-performing methods was supported by performance generated PCA and heatmap clusters that gave insight into target difficulties and the most successful state-of-the-art structure prediction methodologies.

Domain classifications are a useful resource for computational analysis of the protein structure, but elements of their composition are often opaque to potential users. We perform a comparative analysis of our classification ECOD against the SCOPe, SCOP2, and CATH domain classifications with respect to their constituent domain boundaries and hierarchal organization. The coverage of these domain classifications with respect to ECOD and to the PDB was assessed by structure and by sequence. We also conducted domain pair analysis to determine broad differences in hierarchy between domains shared by ECOD and other classifications. Finally, we present domains from the major facilitator superfamily (MFS) of transporter proteins and provide evidence that supports their split into domains and for multiple conformations within these families. We find that the ECOD and CATH provide the most extensive structural coverage of the PDB. ECOD and SCOPe have the most consistent domain boundary conditions, whereas CATH and SCOP2 both differ significantly.

Recent advances in molecular genetics have expanded our knowledge of renal tumors and enabled better classification. These studies have revealed that renal tumors with predominantly “eosinophilic/ oncocytic” cytoplasm include a number of novel biologic subtypes beyond the traditionally well recognized renal oncocytoma (RO) and eosinophilic variant of chromophobe renal cell carcinoma (ChRCC). Herein, we present a comprehensive review of Eosinophilic Vacuolated Tumor (EVT), a recently described novel renal epithelial tumor subtype. Leveraging the comprehensive study of radiology, histopathology, electron microscopy and next generation sequencing of a patient with EVT with sporadic MTOR mutation, we shed insight on the pathogenesis of EVT. These data allows us to postulate that loss of chromosome 1 loss observed in EVT with MTOR mutation may be related to mTORC1 being a dimer since a heterodimer of wild-type and mTOR p.L2427R proteins may not confer sufficient mTORC1 activation. mTORC1 is best known for its role in promoting protein translation. Consistently, dilated cisterns of rough endoplasmic reticulum (ER) were noted on electron microscopic examination that likely corresponded to the cytoplasmic vacuoles seen by light microscopy. This ER expansion may contribute to the increase in phospho-S6 observed in these tumors, as S6 is a ribosomal protein and ribosomes may be increased with the ER expansion. We further discuss the evolving concepts in the classification of this emerging entity.

Centuries of zoological studies have amassed billions of specimens in collections worldwide. Genomics of these specimens promises to reinvigorate biodiversity research. However, because DNA degrades with age in historical specimens, it is a challenge to obtain genomic data for them and analyze degraded genomes. We developed experimental and computational protocols to overcome these challenges and applied our methods to resolve a series of long-standing controversies involving a group of butterflies. We deduced the geographical origins of several historical specimens of uncertain provenance that are at the heart of these debates. Here, genomics tackles one of the greatest problems in zoology: countless old specimens that serve as irreplaceable embodiments of species concepts cannot be confidently assigned to extant species or population due to the lack of diagnostic morphological features and clear documentation of the collection locality. The ability to determine where they were collected will resolve many on-going disputes. More broadly, we show the utility of applying genomics to historical museum specimens to delineate the boundaries of species and populations, and to hypothesize about genotypic determinants of phenotypic traits.

De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.

Allosteric regulation of protein functions is ubiquitous in organismal biology, but the principles governing its evolution are not well understood. Here we discuss recent studies supporting the large-scale existence of latent allostery in ancestor proteins of superfamilies. As suggested, the evolution of allostery could be driven by the need for specificity in paralogs of slow evolving protein complexes with conserved active sites. The same slow evolution is displayed by purifying selection exhibited in allosteric proteins with somatic mutations involved in cancer, where disease-associated mutations are enriched in both orthosteric and allosteric sites. Consequently, disease-associated variants can be used to identify druggable allosteric sites that are specific for paralogs in protein superfamilies with otherwise similar functions.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K(2) subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

Bacterial signal transduction systems sense changes in the environment and transmit these signals to control cellular responses. The simplest one-component signal transduction systems include an input sensor domain and an output response domain encoded in a single protein chain. Alternately, two-component signal transduction systems transmit signals by phosphorelay between input and output domains from separate proteins. The membrane tethered periplasmic bile acid sensor that activates the Vibrio parahaemolyticus type III secretion system adopts an obligate heterodimer of two proteins encoded by partially overlapping VtrA and VtrC genes. This co-component signal transduction system binds bile acid using a lipocalin-like domain in VtrC and transmits the signal through the membrane to a cytoplasmic DNA-binding transcription factor in VtrA. Using the domain and operon organization of VtrA/VtrC, we identify a fast-evolving superfamily of co-component systems in enteric bacteria. Accurate machine learning-based fold predictions for the candidate co-components support their homology in the twilight zone of rapidly evolving sequence and provide mechanistic hypotheses about previously unrecognized lipid-sensing functions.Significance statementUsing the domain and operon organization of VtrA/VtrC, combined with fold predictions, we identify new co-component signal transduction systems in enteric bacteria that likely regulate virulence. We observe that the heterodimeric VtrA/VtrC periplasmic bile acid receptor controlling Vibrio parahaemolyticus T3SS2 is a distant homolog of the ToxR/ToxS master regulator of virulence and has evolved beyond confident sequence recognition. Exploiting the newly developed machine learning methods for structure prediction, we observe a VtrC-like lipocalin fold for both the ToxS periplasmic domain and for other detected periplasmic sensor components. This structure prediction supports the divergent evolution of VtrA/VtrC-like co-component signal transduction systems and suggests a role for lipid sensing in regulating virulence in enteric bacteria.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis. In the fungal pathogen Cryptococcus neoformans, Bim1 is a copper-binding lytic polysaccharide monooxygenase-like protein that participates in copper uptake in concert with the Ctr1 importer to drive virulence mechanisms during fungal meningitis.

This paper reports the evaluation of predictions for the ``CALM1'' challenge in the fifth round of the Critical Assessment of Genome Interpretation held in 2018. In the challenge, the participants were asked to predict effects on yeast growth caused by missense variants of human calmodulin, a highly conserved protein in eukaryotic cells sensing calcium concentration. The performance of predictors implementing different algorithms and methods is similar. Most predictors are able to identify the deleterious or tolerated variants with modest accuracy, with a baseline predictor based purely on sequence conservation slightly outperforming the submitted predictions. Nevertheless, we think that the accuracy of predictions remains far from satisfactory, and the field awaits substantial improvements. The most poorly predicted variants in this round surround functional CALM1 sites that bind calcium or peptide, which suggests that better incorporation of structural analysis may help improve predictions.

Protein target structures for the Critical Assessment of Structure Prediction round 13 (CASP13) were split into evaluation units (EUs) based on their structural domains, the domain organization of available templates, and the performance of servers on whole targets compared to split target domains. Eighty targets were split into 112 EUs. The EUs were classified into categories suitable for assessment of high accuracy modeling (or template-based modeling [TBM]) and topology (or free modeling [FM]) based on target difficulty. Assignment into assessment categories considered the following criteria: (a) the evolutionary relationship of target domains to existing fold space as defined by the Evolutionary Classification of Protein Domains (ECOD) database; (b) the clustering of target domains using eight objective sequence, structure, and performance measures; and (c) the placement of target domains in a scatter plot of target difficulty against server performance used in the previous CASP. Generally, target domains with good server predictions had close template homologs and were classified as TBM. Alternately, targets with poor server predictions represent a mixture of fast evolving homologs, structure analogs, and new folds, and were classified as FM or FM/TBM overlap.

The manual classification of protein domains is approaching its 20th anniversary. ECOD is our mixed manual-automatic domain classification. Over time, the types of proteins which require manual curation has changed. Depositions with complex multidomain and multichain arrangements are commonplace. Transmembrane domains are regularly classified. Repeatedly, domains which are initially believed to be novel are found to have homologous links to existing classified domains. Here we present a brief summary of recent manual curation efforts in ECOD generally combined with specific case studies of transmembrane and multidomain proteins wherein manual curation was useful for discovering new homologous relationships. We present a new taxonomy for the classification of ABC transporter transmembrane domains. We examine alternate topologies of the leucine-specific (LS) domain of Leucine tRNA-synthetase. Finally, we elaborate on a distant homologous links between two helical dimerization domains.

Adopting a proper topology is crucial for transmembrane proteins to perform their functions. We previously reported that ceramide regulates a transmembrane protein called TM4SF20 (transmembrane 4 L six family member 20) through topological inversion by altering the direction through which the protein is translocated across membranes during translation. This regulatory mechanism, denoted regulated alternative translocation (RAT), depends on a GXXXN motif present in the first transmembrane helix of TM4SF20. Here, using site-directed mutagenesis, we show that Asn-26 in the motif is crucial for RAT of TM4SF20, as it cannot be replaced even by Gln. In contrast, Gly-22 in the motif could be substituted by other small residues such as Ala and Ser without affecting RAT of TM4SF20. We further demonstrate that the GXXXN motif alone is insufficient to induce RAT of a transmembrane protein because TM4SF4, a relative of TM4SF20 that also contains the motif in the first transmembrane helix, did not undergo RAT. Using TM4SF40–TM4SF20 chimeras, we identified Pro-29 of TM4SF20 as another important element required for RAT of the protein. Substituting Pro-29 alone did not affect RAT of TM4SF20, whereas replacing Pro-29 together with either Leu-25 or Val-17 of TM4SF20 with the corresponding residues of TM4SF4 abolished RAT of TM4SF20. Because Val-17, Gly-22, Leu-25, Asn-26, and Pro-29 are predicted to reside along the same surface of the transmembrane helix, our results suggest that interactions with other proteins mediated by this surface during translocation may be critical for RAT of TM4SF20.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein–lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro. We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

Bacterial phytopathogens deliver effector proteins into host cells as key virulence weapons to cause disease. Extensive studies revealed diverse functions and biochemical properties of different effector proteins from pathogens. In this study, we show that the Pseudomonas syringae effector AvrE, the founding member of a broadly conserved and pathologically important bacterial effector family, binds to phosphatidylinositides (PIPs) in vitro and shares some properties with eukaryotic PROPPINs (β-propellers that bind polyphosphoinositides). In planta pull down experiments with transgenic Arabidopsis plants expressing AvrE revealed that AvrE is associated with several plant proteins including plasma membrane lipid-raft proteins. These results shed new light on the properties of a bacterial effector that is crucial for bacterial virulence in plants.

DeepMind presented remarkably accurate protein structure predictions at the CASP14 conference. We explored network architectures incorporating related ideas and obtained the best performance with a 3-track network in which information at the 1D sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The 3-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables rapid solution of challenging X-ray crystallography and cryo-EM structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate models of protein-protein complexes from sequence information alone, short circuiting traditional approaches which require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.One-Sentence SummaryAccurate protein structure modeling enables rapid solution of structure determination problems and provides insights into biological function.

Vibrio parahaemolyticus is a seafood-borne pathogen that encodes two type 3 secretion systems (T3SS). The first system, T3SS1, is thought to be maintained in all strains of V. parahaemolyticus to maintain survival in the environment, whereas the second system, T3SS2, is linked to clinical isolates and disease in humans., Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (N. M. Alto and K. Orth, Cold Spring Harbor Perspect Biol 4:a006114, 2012, https://doi.org/10.1101/cshperspect.a006114). The type III secretion system 1 of the marine pathogen Vibrio parahaemolyticus utilizes the sequential action of four effectors to induce a rapid, proinflammatory cell death uniquely characterized by a prosurvival host transcriptional response (D. L. Burdette, M. L. Yarbrough, A Orvedahl, C. J. Gilpin, and K. Orth, Proc Natl Acad Sci USA 105:12497–12502, 2008, https://doi.org/10.1073/pnas.0802773105; N. J. De Nisco, M. Kanchwala, P. Li, J. Fernandez, C. Xing, and K. Orth, Sci Signal 10:eaa14501, 2017, https://doi.org/10.1126/scisignal.aal4501). Herein, we show that this prosurvival response is caused by the action of the channel-forming effector VopQ that targets the host V-ATPase, resulting in lysosomal deacidification and inhibition of lysosome-autophagosome fusion. Recent structural studies have shown how VopQ interacts with the V-ATPase and, while in the ER, a V-ATPase assembly intermediate can interact with VopQ, causing a disruption in membrane integrity. Additionally, we observed that VopQ-mediated disruption of the V-ATPase activates the IRE1 branch of the unfolded protein response (UPR), resulting in an IRE1-dependent activation of ERK1/2 MAPK signaling. We also find that this early VopQ-dependent induction of ERK1/2 phosphorylation is terminated by the VopS-mediated inhibitory AMPylation of Rho GTPase signaling. Since VopS dampens VopQ-induced IRE1-dependent ERK1/2 activation, we propose that IRE1 activates ERK1/2 phosphorylation at or above the level of Rho GTPases. This study illustrates how temporally induced effectors can work as in tandem as agonist/antagonist to manipulate host signaling and reveals new connections between V-ATPase function, UPR, and MAPK signaling. IMPORTANCE Vibrio parahaemolyticus is a seafood-borne pathogen that encodes two type 3 secretion systems (T3SS). The first system, T3SS1, is thought to be maintained in all strains of V. parahaemolyticus to maintain survival in the environment, whereas the second system, T3SS2, is linked to clinical isolates and disease in humans. Here, we found that first system targets evolutionarily conserved signaling systems to manipulate host cells, eventually causing a rapid, orchestrated cells death within 3 h. We have found that the T3SS1 injects virulence factors that temporally manipulate host signaling. Within the first hour of infection, the effector VopQ acts first by activating host survival signals while diminishing the host cell apoptotic machinery. Less than an hour later, another effector, VopS, reverses activation and inhibition of these signaling systems, ultimately leading to death of the host cell. This work provides example of how pathogens have evolved to manipulate the interplay between T3SS effectors to regulate host signaling pathways.

Magnaporthe oryzae causes rice blast disease that threatens global food security by resulting in the severe loss of rice production every year. A tightly regulated life cycle allows M. oryzae to disarm the host plant immune system during its biotrophic stage before triggering plant cell death in its necrotrophic stage., Rice blast disease caused by Magnaporthe oryzae is a devastating disease of cultivated rice worldwide. Infections by this fungus lead to a significant reduction in rice yields and threats to food security. To gain better insight into growth and cell death in M. oryzae during infection, we characterized two predicted M. oryzae metacaspase proteins, MoMca1 and MoMca2. These proteins appear to be functionally redundant and can complement the yeast Yca1 homologue. Biochemical analysis revealed that M. oryzae metacaspases exhibited Ca2+-dependent caspase activity in vitro. Deletion of both MoMca1 and MoMca2 in M. oryzae resulted in reduced sporulation, delay in conidial germination, and attenuation of disease severity. In addition, the double ΔMomca1mca2 mutant strain showed increased radial growth in the presence of oxidative stress. Interestingly, the ΔMomca1mca2 strain showed an increased accumulation of insoluble aggregates compared to the wild-type strain during vegetative growth. Our findings suggest that MoMca1 and MoMca2 promote the clearance of insoluble aggregates in M. oryzae, demonstrating the important role these metacaspases have in fungal protein homeostasis. Furthermore, these metacaspase proteins may play additional roles, like in regulating stress responses, that would help maintain the fitness of fungal cells required for host infection.

BackgroundIn the model bumble bee speciesB. terrestris, both males and females exhibit black coloration on the third thoracic and first metasomal segments. We discovered a fortuitous lab-generated mutant in which this typical black coloration is replaced by yellow. As this same color variant is found in several sister lineages toB. terrestriswithin theBombus s.s. subgenus, this could be a result of ancestral allele sorting.ResultsUtilizing a combination of RAD-Seq and whole-genome re-sequencing approaches, we localized the color-generating variant to a single SNP in the protein-coding sequence of a homeobox transcription factor,cut. Sanger sequencing confirmed fixation of this SNP between wildtype and yellow mutants. Protein domain analysis revealed this SNP to generate an amino acid change (Ala38Pro) that modifies the conformation of coiled-coil structural elements which lie outside the characteristic DNA binding domains. We found all Hymenopterans includingB. terrestrissister lineages possess the non-mutant allele, indicating different mechanism(s) are involved in the same black to yellow transition in nature.ConclusionsCutis a highly pleiotropic gene important for multiple facets of development, yet this mutation generated no noticeable external phenotypic effects outside of setal characteristics. Reproductive capacity was observed to be reduced, however, with queens being less likely to mate and produce female offspring, in a manner similar to workers. Our research implicates a novel developmental player in pigmentation, and potentially caste as well, thus contributing to a better understanding of the evolution of diversity in both of these processes.

Vacuolar-type ATPase (V-ATPase) is a rotary protein pump involved in proton translocation across various cellular membranes using the energy of ATP hydrolysis. Despite previous studies on bacterial and eukaryotic V-ATPases, information on the intact structure of a eukaryotic V-ATPase is missing. Here we report cryo-EM structures of the intact yeast V-ATPase and this complex bound to a bacterial effector. We reveal the interaction of the elusive regulatory subunit H with its neighboring subunits. Insight for the catalysis mechanism is gained by determining conformations of the catalytic subunits either empty or bound with nucleotides.

Viruses are the most abundant life form and infect practically all organisms. Consequently, these obligate parasites are a major cause of human suffering and economic loss. Rossmann-like fold is the most populated fold among α/β-folds in the Protein Data Bank and proteins containing Rossmann-like fold constitute 22% of all known proteins 3D structures. Thus, analysis of viral proteins containing Rossmann-like domains could provide an understanding of viral biology and evolution as well as could propose possible targets for antiviral therapy. We provide functional and evolutionary analysis of viral proteins containing a Rossmann-like fold found in the evolutionary classification of protein domains (ECOD) database developed in our lab. We identified 81 protein families of bacterial, archeal, and eukaryotic viruses in light of their evolution-based ECOD classification and Pfam taxonomy. We defined their functional significance using enzymatic EC number assignments as well as domain-level family annotations.

ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against breast cancers harboring HER2 activating mutations, suggesting these tumors depend on HER2 signaling. Co-occurring HER2 and HER3 (ERBB3) mutations have been reported in patients who respond to neratinib (Hanker et al., Cancer Discov. 2017) suggesting the possibility of cooperativity of both oncogenes. Co-expression of the mutant intracellular domains of HER2 and HER3 in HEK293 cells enhanced phosphorylation of HER3 and ERK compared to expression of either mutant alone, which was blocked by 100 nM neratinib. Interrogation of TCGA, METABRIC, Project GENIE, and Foundation Medicine datasets revealed that gain-of-function mutations in ERBB2 and ERBB3 co-occur with a statistically significant frequency. For example, in GENIE, ERBB2 mutations co-occur with mutations in ERBB3 (8.3% of ERBB2-mutant vs 2.3% of ERBB2 WT; q=1.37x10-10). We hypothesized that co-occurring mutations in HER2 and HER3 cooperate to enhance HER2 signaling and dependence and breast cancer progression. Thirty-four unique breast cancers were found to harbor co-occurring mutations in HER2 and HER3, the most common of which were ERBB2L755S/ERBB3E928G (n=10), ERBB2V777L/ERBB3E928G(n=6), and ERBB2L869R/Q/ERBB3E928G (n=4). Using co-immunoprecipitation assays with HER2 and HER3 antibodies in transfected HEK293 cells, we found that co-expression of HER3E928G with wild type (WT) HER2, or co-expression of HER2L755S or HER2L869R with HER3WT, slightly increased HER2-HER3 dimerization. However, binding was strongest between double mutants. This was accompanied by the highest levels of Y1289 p-HER3 in cells expressing both HER3E928G and each HER2L755S, HER2V777L, or HER2L869R compared to cells expressing each HER2 or HER3 mutant with a respective WT heterodimer partner. Structural modeling of the HER2L869R/HER3E928G double-mutant predicted that the HER3 mutation, located at the dimer interface, may enhance heterodimerization of the kinase domains through decreased bulk and electrostatic repulsion. We also noted that the HER2L755S mutation is predicted to be in close proximity to HER3E928G ( MCF7 “knock-in” cells incorporating HER2L755S, HER2V777L, or HER2L869R (or HER2WT) were stably transduced with HER3E928G or HER3WT. Co-expression of double mutants strongly enhanced estrogen-independent growth in 3D Matrigel over cells expressing either mutant alone. We are currently testing inhibitors of HER2/HER3 signaling, including neratinib ± trastuzumab, trastuzumab + pertuzumab, and the ERBB1-3 antibody mixture Sym013, to determine therapeutic strategies to block the cooperative growth induced by co-occurring HER2 and HER2 mutations. Conclusions: Co-expression of mutant HER2 and mutant HER3 promotes HER2/HER binding, HER3 phosphorylation, and breast tumor cell proliferation. We aim to identify therapeutic vulnerabilities for patients with co-occurring HER2 and HER3 mutations. Citation Format: Hanker AB, Koch JP, Ye D, Sliwoski G, Sheehan J, Kinch LN, Red Brewer M, He J, Miller VA, Lalani AS, Cutler, Jr. RE, Croessmann S, Zabransky DJ, Meiler J, Arteaga CL. Co-occurring gain-of-function mutations in HER2 and HER3 cooperate to enhance HER2/HER3 binding, HER-dependent signaling, and breast cancer growth [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-05.