Protein Family Research Articles

STAT6 promotes innate immunity against BEFV and VSV by inhibiting STUB1 and NIX-mediated MAVS degradation

Signal transducers and activators of transcription 6 (STAT6), an essential member of the STAT protein family, plays vital roles in innate immunity, however, its function in regulating innate immunity through the degradation of MAVS has not been described. In this study, we found that STAT6 suppresses the replication of both bovine ephemeral fever virus (BEFV) and vesicular stomatitis virus (VSV). Further investigations revealed that STAT6 promotes the type I IFN (IFN-I) signaling pathway in the context of BEFV and VSV infection. Moreover, the knockout of STAT6 leads to the degradation of MAVS through both the ubiquitin-proteasome and autophagolysosomal pathways. Mechanistically, STAT6 results in the downregulation of E3 ubiquitin ligase STIP1 homology and Ubox-containing protein 1 (STUB1), inhibits the interaction between STUB1 and MAVS, and reduces STUB1- mediated K48-linked MAVS ubiquitination, thereby inhibiting the MAVS degradation through the ubiquitin-proteasome pathway. Furthermore, STAT6 also suppresses MAVS degradation through the autophagy receptor Bcl2 interacting protein 3 like (NIX)-mediated autophagy pathway. Taken together, our study unveils a novel mechanism by which STAT6 acts as a positive regulator of the type I IFN signaling pathway during BEFV and VSV infection, predominantly by inhibiting MAVS degradation and ultimately suppressing BEFV and VSV infection. These findings provide valuable insights into the regulation of MAVS degradation by STAT6, which may serve as a basis for the design of novel antiviral agents.

HMGB2 promotes smooth muscle cell proliferation through PPAR-γ/PGC-1α pathway-mediated glucose changes in aortic dissection

Background and aimsAortic dissection (AD) is a fatal condition with a complicated pathogenesis. High mobility group protein B2 (HMGB2) is a member of the high mobility group protein family; HMGB2 is involved in innate immunity and inflammatory diseases, but its role in AD remains unclear. MethodsHMGB2-/- mice were generated and treated with β-aminopropionitrile and angiotensin II (Ang II) to an establish AD model. An F12 gel containing AAV9-HMGB2 was applied to overexpress HMGB2 in mice. Pathological changes in the aorta were assessed by visualizing vascular collagen deposition and elastic fiber fracture via H&E, Masson and EVG staining. HMGB2 expression was measured by Western blotting and immunohistochemistry. The MTS, CCK-8 and EdU assays were used to test cell proliferation. ResultsHMGB2 expression was increased in samples from AD patients, samples from AD model mice and human aortic smooth muscle cells (HASMCs). HMGB2 promoted HASMC proliferation. Immunofluorescence staining and plasma membrane protein isolation revealed that HMGB2 decreased GLUT1 expression and promoted GLUT4 translocation. HMGB2 was also found to inhibit the expression of SIRT1/PGC-1α, but blocking the PPAR-γ pathway attenuated this effect. HMGB2-/- significantly reduced the incidence and mortality rates of AD, whereas treatment with AAV9-HMGB2 exacerbated AD. ConclusionsThis study suggested that HMGB2 promotes HASMC proliferation and vascular remodeling by regulating glucose metabolism through the PPAR-γ/SIRT1/PGC-1α pathway. HMGB2 knockdown reduced, whereas HMGB2 overexpression promoted, the occurrence of AD in mice. This study may help elucidate the underlying mechanisms and provide a new preventive target for AD.

Learning protein language contrastive models with multi-knowledge representation

Protein representation learning plays a crucial role in obtaining a comprehensive understanding of biological regulatory mechanisms and in developing proteins and drugs for therapeutic purposes. However, labeled proteins, such as sequenced and functionally annotated data, are incomplete and few. Thus, contrastive learning has emerged as the preferred technique for learning meaningful representations from unlabeled data samples. In addition, at present, natural proteins cannot be fully described by extracting protein knowledge from a single domain. Therefore, Pro-CoRL, a protein contrastive models framework based on multi-knowledge representation learning, was proposed in this study. In particular, Pro-CoRL smooths the objective function using convex approximation, thereby improving the stability of training. Extensive experiments on predicting protein–protein interaction types and clustering protein families have confirmed the high accuracy and robustness of Pro-CoRL.

Two plant membrane-shaping reticulon-like proteins play contrasting complex roles in turnip mosaic virus infection.

Positive-sense RNA viruses remodel cellular cytoplasmic membranes as the membranous sources for the formation of viral replication organelles (VROs) for viral genome replication. In plants, they traffic through plasmodesmata (PD), plasma membrane-lined pores enabling cytoplasmic connections between cells for intercellular movement and systemic infection. In this study, we employed turnip mosaic virus (TuMV), a plant RNA virus to investigate the involvement of RTNLB3 and RTNLB6, two ER (endoplasmic reticulum) membrane-bending, PD-located reticulon-like (RTNL) non-metazoan group B proteins (RTNLBs) in viral infection. We show that RTNLB3 interacts with TuMV 6K2 integral membrane protein and RTNLB6 binds to TuMV coat protein (CP). Knockdown of RTNLB3 promoted viral infection, whereas downregulation of RTNLB6 restricted viral infection, suggesting that these two RTNLs play contrasting roles in TuMV infection. We further demonstrate that RTNLB3 targets the α-helix motif 42LRKSM46 of 6K2 to interrupt 6K2 self-interactions and compromise 6K2-induced VRO formation. Moreover, overexpression of AtRTNLB3 apparently promoted the selective degradation of the ER and ER-associated protein calnexin, but not 6K2. Intriguingly, mutation of the α-helix motif of 6K2 that is required for induction of VROs severely affected 6K2 stability and abolished TuMV infection. Thus, RTNLB3 attenuates TuMV replication, probably through the suppression of 6K2 function. We also show that RTNLB6 promotes viral intercellular movement but does not affect viral replication. Therefore, the proviral role of RTNLB6 is probably by enhancing viral cell-to-cell trafficking. Taken together, our data demonstrate that RTNL family proteins may play diverse complex, even opposite, roles in viral infection in plants.

Single-cell transcriptome profiles and E−64 inhibitor data reveal the essential role of cysteine proteases in the ontogeny of Ichthyophthirius multifiliis

Ichthyophthiriasis (Ich), also known as white spot disease, causes significant economic losses to fish farmers once an outbreak occurs. For fish survival, it is therefore crucial to understand the pathogenic mechanism and find effective prevention methods. In this study, we obtained data for four stages (theront, trophont, protomont and tomont) of Ichthyophthirius multifiliis by single-cell RNA sequencing (scRNA-seq). We found that the invasion-related proteins encoded by highly expressed genes in the theront stage mainly belong to the leishmanolysin family proteins, heat shock proteins, transmembrane proteins and cysteine proteases (CPs). Additionally, the exosome pathway appears to play a significant role in the invasion process of the theront. Since cysteine proteases are expressed at all stages of the I. multifiliis, and five CP-related genes were significantly upregulated at the theront stage of its life cycle — two of which are enriched in the exosome pathway — we incubated I. multifiliis theronts and protomonts with cysteine protease inhibitor (E−64). Our findings revealed that E−64 could kill both stages of the parasite in vitro and affected tomont division and subsequent release. Furthermore, infection experiment showed that E−64 could significantly inhibit the invasion of theronts. Based on our preliminary analysis from the transcriptomic and E−64 experiments, we have confirmed that CPs play a crucial role in I. multifiliis. This research establishes a foundation for future strategies in the prevention and control of Ich.

Insights into the distinct membrane targeting mechanisms of WDR91 family proteins.

WDR91 and SORF1, members of the WD repeat-containing protein 91 family, control phosphoinositide conversion by inhibiting phosphatidylinositol 3-kinase activity on endosomes, which promotes endosome maturation. Here, we report the crystal structure of the human WDR91 WD40 domain complexed with Rab7 that has an unusual interface at the C-terminus of the Rab7 switch II region. WDR91 is highly selective for Rab7 among the tested GTPases. A LIS1 homology (LisH) motif within the WDR91 N-terminal domain (NTD) mediates self-association and may contribute partly to the augmented interaction between full-length WDR91 and Rab7. Both the Rab7 binding site and the LisH motif are indispensable for WDR91 function in endocytic trafficking. For the WDR91 orthologue SORF1 lacking the C-terminal WD40 domain, a C-terminal amphipathic helix (AH) mediates strong interactions with liposomes containing acidic lipids. During evolution the human WDR91 ancestor gene might have acquired a WD40 domain to replace the AH for endosomal membrane targeting.

Unveiling Insights into the Whole Genome Sequencing of Mycobacterium spp. Isolated from Siamese Fighting Fish (Betta splendens)

This study aims to genomically elucidate six isolates of rapidly growing non-tuberculous mycobacteria (RGM) derived from Siamese fighting fish (Betta splendens). These isolates had previously undergone phenotypic and biochemical characterization, antibiotic susceptibility testing, and in vivo virulence assessment. Initial DNA barcoding using the 16S rRNA sequence assigned these six isolates to five different species, namely Mycobacterium chelonae (BN1983), M. cosmeticum (BN1984 and N041), M. farcinogenes (SNSK5), M. mucogenicum (BN1956), and M. senegalense (BN1985). However, the identification relied solely on the highest percent identity of the 16S rRNA gene, raising concerns about the taxonomic ambiguity of these species. Comprehensive whole genome sequencing (WGS) and extended genomic comparisons using multilocus sequence typing (MLST), average nucleotide identity (ANI), and digital DNA–DNA hybridization (dDDH) led to the reclassification of BN1985 and SNSK5 as M. conceptionense while confirming BN1983 as M. chelonae and BN1984 and N041 as M. cosmeticum. Notably, the analysis of the BN1956 isolate revealed a potential new species that is proposed here as M. mucogenicum subsp. phocaicum sp. nov. Common genes encoding “mycobacterial” virulence proteins, such as PE and PPE family proteins, MCE, and YrbE proteins, were detected in all six isolates. Two species, namely M. chelonae and M. cosmeticum, appear to have horizontally acquired T6SS-II (clpB), catalase (katA), GroEL (groel), and capsule (rmlb) from distantly related environmental bacteria such as Klebsiella sp., Neisseria sp., Clostridium sp., and Streptococcus sp. This study provides the first draft genome sequence of RGM isolates currently circulating in B. splendens and underscores the necessity of WGS for the identification and classification of mycobacterial species.

Structure-guided discovery of bile acid derivatives for treating liver diseases without causing itch.

Chronic itch is a debilitating symptom profoundly impacting the quality of life in patients with liver diseases like cholestasis. Activation of the human G-protein coupled receptor, MRGPRX4 (hX4), by bile acids (BAs) is implicated in promoting cholestasis itch. However, the detailed underlying mechanisms remain elusive. Here, we identified 3-sulfated BAs that are elevated in cholestatic patients with itch symptoms. We solved the cryo-EM structure of hX4-Gq in a complex with 3-phosphated deoxycholic acid (DCA-3P), a mimic of the endogenous 3-sulfated deoxycholic acid (DCA-3S). This structure revealed an unprecedented ligand-binding pocket in MRGPR family proteins, highlighting the crucial role of the 3-hydroxyl (3-OH) group on BAs in activating hX4. Guided by this structural information, we designed and developed compound 7 (C7), a BA derivative lacking the 3-OH. Notably, C7 effectively alleviates hepatic injury and fibrosis in liver disease models while significantly mitigating the itch side effects.

Design and Synthesis of a Novel Pyrazole-Based Molecule with Potential Anticancer and Antimicrobial effects: A Multifaceted in silico Approach

Although cancer remains the largest cause of morbidity and mortality globally, there is growing concern about the usage of cancer drugs due to their serious side effects. Therefore, a compound, 3,5-di(1H-indol-6-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide, was synthesized using a mixture of (E)-1, 3-Di(1H-indol-3-yl) prop-2-en-1-one, semicarbazide hydrochloride, and sodium acetate. It was then examined for its anticancer and antimicrobial properties using molecular docking, molecular dynamic simulations and molecular mechanics, and generalised born surface area (MM/GBSA). The molecular docking was performed by targeting the cancer and antimicrobial associated proteins, namely protein tyrosine phosphatase L1 (1WCH), tankyrase-1 (5ETY), and phosphotransferase enzyme family protein (6SUI). Eventually, 3,5-di(1H-indol-6-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide was found to be the most promising drug candidate for fighting both cancer and microbial infections. With docking scores of -9.730 Kcal/mol for 1WCH, -9.142 Kcal/mol for 5ETY, and -7.026 Kcal/mol for 6SUI, it showed strong interactions with all the target proteins. To investigate the binding stability of 3,5-di(1H-indol-6-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide with the target molecules, molecular dynamics simulations were performed for 100 ns on the docked complexes. With an RMSD of less than 2.8Å, the synthesized compound was highly stable with all of the targets for 100 ns. Furthermore, the evaluation of the binding free energies (MM/GBSA) of these complexes revealed that the simulated environment had no significant influence on the binding energies, implying the synthesized molecule has strong binding affinities with the target proteins. Furthermore, in the toxicity assessment, the active probability ratio for 3,5-di(1H-indol-6-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide was less than 0.60, indicating a low probability of organ toxicity and endpoint toxicity. Based on the results, the research concludes that the synthesized novel pyrazole-based compound could be a promising candidate for the development of new therapeutic drugs for the treatment of cancer and microbial infections.

Systematic expression analysis of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein (CAP) superfamily in Arabidopsis.

The Cysteine-rich secretory proteins (CRISPS), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) protein (CAP) superfamily members are found in multiple eukaryotic organisms, including yeasts, animals, and plants. Although one of the plant CAP family genes, PR-1 is known to respond to pathogen infection in plants, the functions of other CAP family genes in Arabidopsis remain largely unknown. In this study, we conducted a comprehensive analysis of the similarities, loci, and expression patterns of 22 Arabidopsis CAP genes/proteins, providing a clue to elucidate their molecular functions. According to the promoter-β-glucuronidase (GUS) analysis, members of the Arabidopsis CAP family were expressed in various young tissues or organs, such as root and shoot meristems, reproductive tissues, and particularly at the lateral root initiation site before the formation of the lateral root primordium, with distinct expression specificity. In particular, CAP51, CAP52, and CAP53 were specifically expressed in the cortical cells at the lateral root developing regions, suggesting that these genes may function in lateral root development. Thus, the expression patterns of Arabidopsis CAP family genes suggest that CAP family proteins may have certain function in the expressed organs or tissues in Arabidopsis plant.

Proapoptotic and antimigration properties of osthole in combination with LY294002 against human glioma cells.

Anaplastic astrocytoma and glioblastoma multiforme are infiltrating and vascularized gliomas with a high degree of chemoresistance and metastasis. Our previous studies have shown that osthole may be of great importance in the treatment of gliomas. Therefore, in this work, for the first time, coumarin was used in combination with LY294002-an inhibitor of the PI3K-Akt/PKB-mTOR pathway, which is overly active in gliomas. MOGGCCM and T98G cells were incubated with osthole and LY294002, alone and in combination. Staining with specific fluorochromes was used to visualize cell death and the scratch test to assess the migration. The level of proteins was estimated by immunoblotting. Forming protrusions were visualized by SEM, and immunocytochemistry was used to determine the localization of proteins. Additionally, the expression of Bcl-2, beclin 1 and Raf kinase was silenced using specific siRNA. The obtained results showed that osthole in combination with LY294092 effectively inhibited the migration of glioma cells by reducing the level of metaloproteinases and Rho family proteins, as well as decreasing the level of N-cadherin. In addition, the combination of compounds induced apoptosis. New combination of compounds shows a high pro-apoptotic potential and also inhibits the migration of gliomas cells.

Application of immunoinformatics to develop a novel and effective multiepitope chimeric vaccine against Variovorax durovernensis

Bloodstream infections pose a significant public health challenge caused by resistant bacteria such as Variovorax durovernensis, a recently reported Gram-negative bacterium, worsening the burden on healthcare systems. The design of a vaccine using chimeric peptides derived from a representative V. durovernensis strain holds significant promise for preventing disease onset. The current study aimed to employ reverse vaccinology (RV) approaches such as the retrieval of V. durovernensis proteomics data, removal of redundant proteins by CD-HIT, filtering of non-homologous proteins to humans and essential proteins, identification of outer membrane (OM) proteins by CELLO and PSORTb. Following these steps immunoinformatic approaches were applied, such as epitope prediction by IEDB, vaccine design using linkers and adjuvant andanalysis of antigenicity, allergenicity, safety and stability. Among the 4208 nonredundant proteins, an OmpA family protein (A0A940EKP4) was designated a potential candidate for the development of a multiepitope vaccine construct. Upon analysis of OM protein, six immunodominant (B cell) epitopes were found on the basis of the chimeric construct following the prediction of CTL stands cytotoxic T lymphocyte and HTL stands helper T lymphocyte epitopes. To ensure comprehensive population coverage globally, the CTL and HTL coverage rates were 58.18% and 46.56%, respectively, and 77.23% overall. By utilizing EAAAK, GPGPG, and AAY linkers, Cholera toxin B subunit adjuvants, and appropriate epitopes were smoothly incorporated into a chimeric vaccine effectively triggering both adaptive and innate immune responses. For example, the administered antigen showed a peak in counts on the fifthday post injection and then gradually declined until the fifteenth day. Elevated levels of several antibodies (IgG + IgM > 700,000; IgM > 600,000; IgG1 + IgG2; IgG1 > 500,000) were observed as decreased in the antigen concentration. Molecular dynamics simulations carried out via iMODS revealed strong correlations between residue pairs, highlighting the stability of the docked complex. The designed vaccine has promising potential in eliciting specific immunogenic responses, thereby facilitating future research for vaccine development against V. durovernensis.

Interference of FZD2 suppresses proliferation, vasculogenic mimicry and stemness in glioma cells via blocking the Notch/NF‑κB signaling pathway.

Frizzled family protein 2 (FZD2) is widely associated with tumor development and metastasis. The present study aimed to gain an insight into the role and regulatory mechanism of FZD2 in glioma. The expression level of FZD2 in normal astrocyte and glioma cells was determined by reverse transcription-quantitative PCR and western blotting, and cell transfection was conducted for FZD2 expression knockdown. Malignant behaviors including cell proliferation, migration and invasion, vasculogenic mimicry (VM) and cell stemness were determined using Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine (EdU) staining, colony formation, wound healing, Transwell, 3D culturing and sphere formation assays. The expression levels of proteins related to stemness, epithelial-mesenchymal transition (EMT) and Notch/NF-κB signaling were measured by western blotting. Then, the Notch agonist, Jagged-1 (JAG), was adopted for rescue experiments. The results demonstrated that FZD2 was highly expressed in glioma cells. Interference of FZD2 expression suppressed the proliferation of glioma cells, as evidenced by the reduced cell viability and the number of EdU+ cells and colonies. Meanwhile, the reduced sphere formation ability and decreased protein expression of Nanog, Sox2 and Oct4 following FZD2 knockdown confirmed that FZD2 repressed cell stemness in glioma. Additionally, FZD2 knockdown suppressed the migration, invasion, EMT and VM formation capabilities of glioma cells, and also blocked the Notch/NF-κB signaling pathway. Furthermore, activation of Notch by JAG treatment partially reversed the aforementioned FZD2 knockdown-mediated changes in glioma cell malignant behaviors. In conclusion, FZD2 may contribute to glioma progression through activating the Notch/NF-κB signaling pathway, providing a plausible therapeutic target for the treatment of glioma.

CDK14 is regulated by IGF2BP2 and involved in osteogenic differentiation via Wnt/β-catenin signaling pathway in vitro

AimsCyclin-dependent kinase (CDK) family proteins involve in various cellular processes via regulating the cell cycle; however, their expression during osteogenic differentiation and postmenopausal osteoporosis remains poorly understood. Main methodsUsing bioinformatics, we screened for CDK14 bound to Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and explored its expression in vitro with time-gradient model and in a mouse model of postmenopausal osteoporosis, building on prior research. Subsequently, we investigated its effect on osteoblast proliferation, cell cycle dynamics, and osteogenic differentiation by administering CDK14 siRNA and the covalent inhibitor FMF-04-159-2. Furthermore, we examined the interaction between IGF2BP2 and CDK14. Finally, we validated the regulatory role of CDK14 on the Wnt/β-catenin pathway. Key findingsOur findings demonstrate a time-dependent CDK14 expression patterns during osteogenic differentiation of MC3T3-E1 cell line, with an initial increase followed by gradual decline over time. Notably, CDK14 expression exhibited significant reduction in bone tissue of postmenopausal osteoporosis mouse model. CDK14 inhibition altered osteoblast cell cycle dynamics, significantly reduced cellular proliferation capacity, and impaired osteogenic differentiation ability. IGF2BP2 interacted with CDK14 mRNA, and stabilizing mRNA's structure and inhibiting its degradation. Additionally, CDK14 facilitated Low-density lipoprotein receptor-related protein 6 (LRP6) and Glycogen synthase kinase 3β (GSK3β) phosphorylation, thus regulating β-catenin levels. SignificanceThese findings provide further insight into the molecular mechanisms governing osteoblast proliferation, differentiation and osteoporosis.

Using reverse vaccinology techniques combined with B-cell epitope prediction to screen potential antigenic proteins of the bovine pathogen Clostridium perfringens type A

Clostridium perfringens type A frequently causes necrohaemorrhagic enteritis in cattle, a rapidly progressing disease with a high mortality rate, thus inflicting substantial economic losses in the cattle industry. Effective prevention and control of this disease rely on rapid detection and vaccination strategies, making the screening of antigenic proteins with diagnostic and vaccine potential particularly crucial. In this study, we conducted a pangenomic analysis of 15 bacterial strains, grounded in traditional reverse vaccinology and supplemented with B-cell linear and conformational epitope analysis tools. This approach led to the identification of 2304 core genes and 3606 accessory genes, among which 58 surface-exposed proteins, encoded by core genes, were identified Proteins lacking tertiary structure information were predicted via AlphaFold2, ultimately identifying four target proteins and 14 candidate proteins enriched with linear and conformational epitopes, including virulence proteins such as alpha-toxin, theta-toxin, and alpha-clostripain, and extracellular solute-binding proteins, rhodanese-like proteins, and the accessory gene-encoded lysozyme inhibitor LprI family protein. Our findings demonstrate that the combined use of multiple B-cell epitope analysis tools can help overcome the limitations of any single tool. The proteins selected in this study offer valuable references for rapid diagnostics and the development of genetically engineered vaccines.

387 (PB375): Tumor adaptation to PI3K inhibition and its stability increases as a function of time and is overcome by combined inhibiting Bcl2 family proteins

387 (PB375): Tumor adaptation to PI3K inhibition and its stability increases as a function of time and is overcome by combined inhibiting Bcl2 family proteins

249 (PB237): The bromodomain and extraterminal domain family proteins, BRD2, BRD3 and BRD4, are enrolled in H19-dependent transcriptional regulation of cell adhesion molecules and modulate metastatic dissemination program in prostate cancer

249 (PB237): The bromodomain and extraterminal domain family proteins, BRD2, BRD3 and BRD4, are enrolled in H19-dependent transcriptional regulation of cell adhesion molecules and modulate metastatic dissemination program in prostate cancer

Grb7, Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism

BackgroundThe growth factor receptor bound protein 7 (Grb7) family of signalling adaptor proteins comprises Grb7, Grb10 and Grb14. Each can interact with the insulin receptor and other receptor tyrosine kinases, where Grb10 and Grb14 inhibit insulin receptor activity. In cell culture studies they mediate functions including cell survival, proliferation, and migration. Mouse knockout (KO) studies have revealed physiological roles for Grb10 and Grb14 in glucose-regulated energy homeostasis. Both Grb10 KO and Grb14 KO mice exhibit increased insulin signalling in peripheral tissues, with increased glucose and insulin sensitivity and a modestly increased ability to clear a glucose load. In addition, Grb10 strongly inhibits fetal growth such that at birth Grb10 KO mice are 30% larger by weight than wild type littermates.ResultsHere, we generate a Grb7 KO mouse model. We show that during fetal development the expression patterns of Grb7 and Grb14 each overlap with that of Grb10. Despite this, Grb7 and Grb14 did not have a major role in influencing fetal growth, either alone or in combination with Grb10. At birth, in most respects both Grb7 KO and Grb14 KO single mutants were indistinguishable from wild type, while Grb7:Grb10 double knockout (DKO) were near identical to Grb10 KO single mutants and Grb10:Grb14 DKO mutants were slightly smaller than Grb10 KO single mutants. In the developing kidney Grb7 had a subtle positive influence on growth. An initial characterisation of Grb7 KO adult mice revealed sexually dimorphic effects on energy homeostasis, with females having a significantly smaller renal white adipose tissue depot and an enhanced ability to clear glucose from the circulation, compared to wild type littermates. Males had elevated fasted glucose levels with a trend towards smaller white adipose depots, without improved glucose clearance.ConclusionsGrb7 and Grb14 do not have significant roles as inhibitors of fetal growth, unlike Grb10, and instead Grb7 may promote growth of the developing kidney. In adulthood, Grb7 contributes subtly to glucose mediated energy homeostasis, raising the possibility of redundancy between all three adaptors in physiological regulation of insulin signalling and glucose handling.

Understanding epistatic networks in the B1 β-lactamases through coevolutionary statistical modeling and deep mutational scanning.

Throughout evolution, protein families undergo substantial sequence divergence while preserving structure and function. Although most mutations are deleterious, evolution can explore sequence space via epistatic networks of intramolecular interactions that alleviate the harmful mutations. However, comprehensive analysis of such epistatic networks across protein families remains limited. Thus, we conduct a family wide analysis of the B1 metallo-β-lactamases, combining experiments (deep mutational scanning, DMS) on two distant homologs (NDM-1 and VIM-2) and computational analyses (in silico DMS based on Direct Coupling Analysis, DCA) of 100 homologs. The methods jointly reveal and quantify prevalent epistasis, as ~1/3rd of equivalent mutations are epistatic in DMS. From DCA, half of the positions have a >6.5 fold difference in effective number of tolerated mutations across the entire family. Notably, both methods locate residues with the strongest epistasis in regions of intermediate residue burial, suggesting a balance of residue packing and mutational freedom in forming epistatic networks. We identify entrenched WT residues between NDM-1 and VIM-2 in DMS, which display statistically distinct behaviors in DCA from non-entrenched residues. Entrenched residues are not easily compensated by changes in single nearby interactions, reinforcing existing findings where a complex epistatic network compounds smaller effects from many interacting residues.

TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44.

The ER is the organelle of nucleated cells that produces lipids, sugars, and proteins. More than 20 ER-resident members of the protein disulfide isomerase (PDI) family regulate formation, isomerization, and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4, and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least-known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multipass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57, and ERp44. The association between TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER localization of TMX5 in pre-Golgi compartments. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1β, Prx4, ERAP1, and SUMF1 that operate in pre-Golgi compartments but lack localization sequences required to position themselves and rely on ERp44 engagement for proper intercompartmental distribution.