Protein-protein Interaction Domains Research Articles

Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

The binding selectivity of the C-terminal SH3 domain of Grb2, but not its folding pathway, is dictated by its contiguous SH2 domain

The adaptor protein Grb2, or Growth factor receptor bound protein 2, possesses a pivotal role in the transmission of fundamental molecular signals in the cell. Despite lacking enzymatic activity, Grb2 functions as a dynamic assembly platform, orchestrating intracellular signals through its modular structure. This study delves into the energetic communication of Grb2 domains, focusing on the folding and binding properties of the C-SH3 domain linked to its neighboring SH2 domain. Surprisingly, while the folding and stability of C-SH3 remain robust and unaffected by SH2 presence, significant differences emerge in the binding properties when considered within the tandem context compared to isolated C-SH3. Through a double mutant cycle analysis, we highlighted a subset of residues, located at the interface with the SH2 domain and far from the binding site, finely regulating the binding of a peptide mimicking a physiological ligand of the C-SH3 domain. Our results have mechanistic implications about the mechanisms of specificity of the C-SH3 domain, indicating that the presence of the SH2 domain optimizes binding to its physiological target, and emphasizing the general importance of considering supramodular multidomain protein structures to understand the functional intricacies of protein-protein interaction domains.

Addressing the Binding Mechanism of the Meprin and TRAF-C Homology Domain of the Speckle-Type POZ Protein Using Protein Engineering.

Protein-protein interactions play crucial roles in a wide range of biological processes, including metabolic pathways, cell cycle progression, signal transduction, and the proteasomal system. For PPIs to fulfill their biological functions, they require the specific recognition of a multitude of interacting partners. In many cases, however, protein-protein interaction domains are capable of binding different partners in the intracellular environment, but they require precise regulation of the binding events in order to exert their function properly and avoid misregulation of important molecular pathways. In this work, we focused on the MATH domain of the E3 Ligase adaptor protein SPOP in order to decipher the molecular features underlying its interaction with two different peptides that mimic its physiological partners: Puc and MacroH2A. By employing stopped-flow kinetic binding experiments, together with extensive site-directed mutagenesis, we addressed the roles of specific residues, some of which, although far from the binding site, govern these transient interactions. Our findings are compatible with a scenario in which the binding of the MATH domain with its substrate is characterized by a fine energetic network that regulates its interactions with different ligands. Results are briefly discussed in the context of previously existing work regarding the MATH domain.

TAZ2 truncation confers overactivation of p300 and cellular vulnerability to HDAC inhibition

The histone acetyltransferase p300/CBP is composed of several conserved domains, among which, the TAZ2 domain is known as a protein-protein interaction domain that binds to E1A and various transcription factors. Here we show that TAZ2 has a HAT autoinhibitory function. Truncating p300/CBP at TAZ2 leads to hyperactive HAT and elevated histone H3K27 and H3K18 acetylation in cells. Mechanistically, TAZ2 cooperates with other HAT neighboring domains to maintain the HAT active site in a ‘closed’ state. Truncating TAZ2 or binding of transcription factors to TAZ2 induces a conformational change that ‘opens’ the active site for substrate acetylation. Importantly, genetic mutations that lead to p300/CBP TAZ2 truncations are found in human cancers, and cells with TAZ2 truncations are vulnerable to histone deacetylase inhibitors. Our study reveals a function of the TAZ2 domain in HAT autoinhibitory regulation and provides a potential therapeutic strategy for the treatment of cancers harboring p300/CBP TAZ2 truncations.

Cryo-EM structure of the chain-elongating E3 ubiquitin ligase UBR5.

UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT domain-containing ubiquitin ligase has recently been identified as an important regulator of oncogenes, e.g., MYC, but little is known about its structure or mechanisms of substrate engagement and ubiquitination. Here, we present the cryo-EM structure of human UBR5, revealing an α-solenoid scaffold with numerous protein-protein interacting motifs, assembled into an antiparallel dimer that adopts further oligomeric states. Using cryo-EM processing tools, we observe the dynamic nature of the UBR5 catalytic domain, which we postulate is important for its enzymatic activity. We characterise the proteasomal nuclear import factor AKIRIN2 as an interacting protein and propose UBR5 as an efficient ubiquitin chain elongator. This preference for ubiquitinated substrates and several distinct domains for protein-protein interactions may explain how UBR5 is linked to several different signalling pathways and cancers. Together, our data expand on the limited knowledge of the structure and function of HECT E3 ligases.

Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners.

SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates. Furthermore, SPOP is responsible for the regulation of both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions. The recognition of SPOP and its physiological partners is mediated by two protein-protein interaction domains. Among them, the MATH domain recognizes different substrates, and it is critical for orchestrating diverse cellular pathways, being mutated in several human diseases. Despite its importance, the mechanism by which the MATH domain recognizes its physiological partners has escaped a detailed experimental characterization. In this work, we present a characterization of the binding mechanism of the MATH domain of SPOP with three peptides mimicking the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN. Furthermore, by taking advantage of site-directed mutagenesis, we address the role of some key residues of MATH in the binding process. Our findings are briefly discussed in the context of previously existing data on the MATH domain.

#3419 LINARIN PROTECTS THE KIDNEY FROM ISCHEMIA-REPERFUSION INJURY AND SUPPRESSES ETS2 INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Ischemia/reperfusion injury (IRI), a participant in acute kidney injury (AKI), can occur as a series of pathological processes such as inflammation. Linarin (LIN) has been widely used for different diseases. To confirm the anti-inflammatory value and relevant mechanism of LIN during IRI, in vivo and vitro model were established. Method LIN or isovolumetric DMSO was given, and histologic analysis, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), serum creatinine (SCr) testing and blood urea nitrogen (BUN) were used to evaluate kidney injury. Microarray analysis, protein-protein interaction (PPI) analysis and molecular docking were used to identify target protein of LIN. Results At first, we find that LIN could inhabit the kidney injury in vivo IRI model and decrease the expression of IL-12 p40 in vitro IRI model. To explore the mechanism of LIN, we collected raw data from public microarray database and identified E26 oncogene homolog 2 (ETS2) as a target protein of LIN according to microarray analysis, PPI and molecular docking. The contact area is highly conserved and located on a protein-protein interaction domain of ETS2 which indicated that LIN may alter the interaction with synergistical protein in the regulation of IL-12 p40 expression. Conclusion Our study demonstrated the anti-inflammatory effect of LIN during IRI-triggered AKI, broadening the medicinal value of LIN and the therapeutic options for IRI-triggered AKI.

Global Discovery of Covalent Modulators of Ribonucleoprotein Granules.

Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein-protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.

Spatio-temporal regulation of endocytic protein assembly by SH3 domains in yeast.

Clathrin-mediated endocytosis is a conserved eukaryotic membrane trafficking pathway that is driven by a sequentially assembled molecular machinery that contains over 60 different proteins. SH3 domains are the most abundant protein-protein interaction domain in this process, but the function of most SH3 domains in protein dynamics remains elusive. Using mutagenesis and live-cell fluorescence microscopy in the budding yeast Saccharomyces cerevisiae, we dissected SH3-mediated regulation of the endocytic pathway. Our data suggest that multiple SH3 domains regulate the actin nucleation-promoting Las17-Vrp1 complex, and that the network of SH3 interactions coordinates both Las17-Vrp1 assembly and dissociation. Furthermore, most endocytic SH3 domain proteins use the SH3 domain for their own recruitment, while a minority use the SH3 domain to recruit other proteins and not themselves. Our results provide a dynamic map of SH3 functions in yeast endocytosis and a framework for SH3 interaction network studies across biology.

WD repeat protein 54-mediator of ErbB2-driven cell motility 1 axis promotes bladder cancer tumorigenesis and metastasis and impairs chemosensitivity

One of the most abundant protein-protein interaction domains in the human proteome is the WD40 repeat (WDR) domain. A Gene Expression Omnibus dataset revealed 37 differentially expressed WDR domain genes in bladder cancer (BC). WD repeat domain 54 (WDR54), an upregulated WDR domain gene, was selected for further investigation. Sixty pairs of frozen BC tumor and non-malignant bladder tissues and 83 paraffin-embedded BC tissue specimens were obtained. Loss-/gain-of-function experiments were carried out using BC and xenograft tumor models. WDR54 was overexpressed in BC cells, and its high expression was linked to tumor stage and lymph node metastases in patients. WDR54 contributed to the tumorigenesis and metastasis of BC and impaired its chemosensitivity. WDR54 prevented the degradation and ubiquitination of the mediator of ErbB2-driven cell motility 1 (MEMO1). WDR54 also promoted the interaction between MEMO1 and insulin receptor substrate 1 (IRS1) and activated the IRS1/AKT/β-catenin pathway in BC cells. Particularly, WDR54 depended on MEMO1 to exert its biological functions. Our study demonstrated the relevance of WDR54 in BC and provides insight into the molecular mechanism underlying BC.

Genome wide analysis of BREVIS RADIX gene family from wheat (Triticum aestivum): A conserved gene family differentially regulated by hormones and abiotic stresses

BREVIS RADIX is a plant specific gene family with unique protein-protein interaction domain. It regulates developmental processes viz. root elongation and tiller angle which are pertinent for crop improvement. In the present study, five BRX family genes were identified in wheat genome and clustered into five sub-groups. Phylogenetic and synteny analyses revealed evolutionary conservation among BRX proteins from monocot species. Expression analyses showed abundance of TaBRXL1 transcripts in vegetative and reproductive tissues except flag leaf. TaBRXL2, TaBRXL3 and TaBRXL4 showed differential, tissue specific and lower level expression as compared to TaBRXL1. TaBRXL5-A expressed exclusively in stamens. TaBRXL1 was upregulated under biotic stresses while TaBRXL2 expression was enhanced under abiotic stresses. TaBRXL2 and TaBRXL3 were upregulated by ABA and IAA in roots. In shoot, TaBRXL2 was upregulated by ABA while TaBRXL3 and TaBRXL4 were upregulated by IAA. Expression levels, tissue specificity and response time under different conditions suggest distinct as well as overlapping functions of TaBRX genes. This was also evident from global co-expression network of these genes. Further, TaBRX proteins exhibited homotypic and heterotypic interactions which corroborated with the role of BRX domain in protein-protein interaction. This study provides leads for functional characterization of TaBRX genes.

Stafiba: A STAT5-Selective Small-Molecule Inhibitor.

The transcription factors STAT5a and STAT5b are constitutively active in many human tumors. Combined inhibition of both STAT5 proteins is a valuable approach with promising applications in tumor biology. We recently reported resorcinol bisphosphate as a moderately active inhibitor of the protein-protein interaction domains, the SH2 domains, of both STAT5a and STAT5b. Here, we describe the development of resorcinol bisphosphate to Stafiba, a phosphatase-stable inhibitor of STAT5a and STAT5b with activity in the low micromolar concentration range. Our data provide insights into the structure-activity relationships of resorcinol bisphosphates and the corresponding bisphosphonates for use as inhibitors of both STAT5a and STAT5b.

A rare case report of AEC syndrome, Ankyloblepharon, ectodermal dysplasia and cleft LIP/cleft palate

The AEC syndrome or Hay wells syndrome is an unusual autosomal dominant disorder characterised by Ankyloblepharon, Ectodermal dysplasia and Cleft palate and/or Cleft lip. This syndrome occurs as a result of missense mutation in TP63 affecting P63 SAM of the gene, which is a protein-protein interaction domain. It is associated with some irregularities like Cleft palate /Cleft lip, severe scalp erosions and abnormalities of epidermal appendages including hypotrichosis, hypodontia, absent or dystrophic nails & mild hypohydrosis. We, here by report a case of full term baby born to third degree consanguineous parents with features of cleft lip and cleft palate, multiple erosions present over back, alopecia over left side of scalp along with few erosions over right side of scalp, sparse eye brows & eye lashes, Ankyloblepharon, Microphallus, Dystrophic finger and toe nails due to its rarity. Diagnosis is made commonly by clinical examination and Genetic analysis.

Templated Generation of a Bcl-xL Inhibitor by Isomer-Free SPAAC Based on Azacyclonon-5-yne.

High-affinity inhibitors of large protein-protein interactions often have a high molecular weight, which compromises their cell permeability and oral bioavailability. We recently presented isomer-free, strain-promoted azide-alkyne cycloaddition (iSPAAC) as a method by which to generate large, chemically uniform bioactive molecules inside living cells from two smaller components with higher cell permeability. Here, we present the synthesis of Fmoc-protected azacyclonon-5-yne (Fmoc-ACN) as the first cyclononyne suitable for iSPAAC. ACN facilitated the structure-guided development of a single-digit micromolar triazole inhibitor of the protein-protein interaction domain of the antiapoptotic protein Bcl-xL . Inhibitor formation in aqueous buffer at 37 °C, templated by the target protein Bcl-xL , proceeded 2800 times faster than the reaction between Fmoc-ACN and benzyl azide under standard conditions in acetonitrile. Our data demonstrate the utility of cyclononynes for iSPAAC and their potential for achieving vastly accelerated templated reactions in aqueous environments.

Resonance assignment and secondary structure of the tandem harmonin homology domains of human RTEL1.

Regulator of telomere elongation helicase 1 (RTEL1) is an Fe-S cluster containing DNA helicase that plays important roles in telomere DNA maintenance, DNA repair, and genomic stability. It is a modular protein comprising an N-terminal helicase domain, two tandem harmonin homology domains 1 & 2 (HHD1 and HHD2), and a C-terminal C4C4 type RING domain. The N-terminal helicase domain disassembles the telomere t/D-loop and unwinds the G-quadruplex via its helicase activity. The C-terminal RING domain interacts with telomere DNA binding protein TRF2 and helps RTEL1 recruitment to the telomere. The tandem HHD1 and HHD2 are characterized as a putative protein-protein interaction domain and have recently been shown to interact with a DNA repair protein SLX4. Several mutations associated with Hoyeraal-Hreidarsson syndrome and pulmonary fibrosis have been found in HHD1 and HHD2 of RTEL1. However, these domains have not been characterized for their structures. We have expressed and purified HHD1 and HHD2 of human RTEL1 for their characterization using solution NMR spectroscopy. Here, we report near complete backbone and sidechain 1H, 13C and 15N chemical shift assignments and secondary structure of the HHD1 and HHD2 domains of human RTEL1.

Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae.

Toll-like receptor (TLR) signaling is key to detect pathogens and initiating inflammation. Ligand recognition triggers the assembly of supramolecular organizing centers (SMOCs) consisting of large complexes composed of multiple subunits. Building such signaling hubs relies on Toll Interleukin-1 Receptor (TIR) and Death Domain (DD) protein-protein interaction domains. We have expressed TIR domain-containing components of the human myddosome (TIRAP and MyD88) and triffosome (TRAM and TRIF) SMOCs in Saccharomyces cerevisiae, as a platform for their study. Interactions between the TLR4 TIR domain, TIRAP, and MyD88 were recapitulated in yeast. Human TIRAP decorated the yeast plasma membrane (PM), except for the bud neck, whereas MyD88 was found at cytoplasmic spots, which were consistent with endoplasmic reticulum (ER)-mitochondria junctions, as evidenced by co-localization with Mmm1 and Mdm34, components of the ER and Mitochondria Encounter Structures (ERMES). The formation of MyD88-TIRAP foci at the yeast PM was reinforced by co-expression of a membrane-bound TLR4 TIR domain. Mutations in essential residues of their TIR domains aborted MyD88 recruitment by TIRAP, but their respective subcellular localizations were unaltered. TRAM and TRIF, however, did not co-localize in yeast. TRAM assembled long PM-bound filaments that were disrupted by co-expression of the TLR4 TIR domain. Our results evidence that the yeast model can be exploited to study the interactions and subcellular localization of human SMOC components in vivo.

Crystal structure of the PDZ4 domain of MAGI2 in complex with PBM of ARMS reveals a canonical PDZ recognition mode

Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI2) is a neuronal scaffold protein that plays critical roles at synaptic junctions by assembling neurotransmitter receptors and cell adhesion proteins through its multiple protein-protein interaction domains, including six PDZ domains, two phosphoserine-phosphothreonine binding WW domains, and a guanylate kinase GK domain. Previous studies showed that MAGI2 participates in formation of tetrameric complexes with PDZ-GEF1, TrkA receptor, and ankyrin repeat-rich membrane spanning (ARMS) protein at late endosomes and is crucial for neurite outgrowth. However, the molecular mechanism governing the assembly of these complexes remains unknown. Here, we characterize the direct interaction between MAGI2 and ARMS through multiple biochemical assays. Moreover, our solved crystal structure of the truncated PDZ4/PBM (PDZ binding motifs) complex of MAGI2 and ARMS proteins (MAGI2-PDZ4/ARMS-PBM) reveals that the binding interface lies between the αB/βB groove from the PDZ4 of MAGI2 and the C-terminal PBM from ARMS. The structure reveals high similarity to others in this protein family where canonical PDZ/PBM interactions are observed. However, the conserved “GLGF” motif in the PSD-95-PDZ3 changes to “GFGF” in the MAGI2-PDZ4/ARMS-PBM complex. We further validated our crystal structure through serial mutagenesis assays. Taken together, our study provides the biochemical details and binding mechanisms that underpin the stabilization of the MAGI2-PDZ4/ARMS-PBM complex, thereby offering a biochemical and structural basis for further understanding of the functional roles of MAGI2, ARMS, PDZ-GEF1, and TrkA in forming the tetrameric receptor complex in neuronal signaling.

Selected Articles from This Issue

BRAF mutation is a common driver event in melanoma, rendering the tumors reliant on constitutive signaling through the MAp. pathway and the extracellular signal-regulated kinases (ERK) 1 and 2. Thus, MAPK pathway inhibitors that act through both ERK1 and ERK2 have become a key component of the therapeutic arsenal against melanoma, but their use is associated with dose-limiting toxicities. Here, Crowe and colleagues demonstrate that BRAF-mutant melanoma is specifically reliant on ERK2 signaling. Targeted ERK2 ablation resulted in transcriptomic and proteomic alterations that aligned with those effected by full MAPK pathway inhibitors, whereas targeted ERK1 ablation produced a distinct set of responses. Both isoforms promoted cell viability, but ERK2 loss suppressed MAPK pathway activity and cell proliferation more effectively than ERK1 loss. Moreover, ERK2 was shown to compensate for ERK1 depletion in the context of BRAF mutation, whereas ERK1 was not able to compensate for loss of ERK2 activity. The authors argue that these findings support the development of ERK2-selective inhibitors, which could potentially be achieved through the targeting of protein-protein interaction domains that are differentially utilized between the two isoforms.Mutation of DNA damage-responsive Fanconi Anemia family members (FANC) is associated with hereditary cancer syndromes and familial cancers, particularly breast and ovarian cancer, due to their role in mediating homologous recombination repair. In particular, FANCJ has been nominated as a key tumor suppressor in several cancer types, though functional data on the many clinically identified FANCJ mutations are lacking. Here, Calvo and colleagues employ a mutation library to screen for loss-of-function (LOF) mutations that confer sensitivity to interstrand crosslinking (ICL) agents. They find that missense and nonsense mutations in the helicase domain are associated with FANCJ LOF, whereas mutations outside this domain did not affect the response to ICL chemotherapeutics. Notably, mutations in the carboxy-terminal domain, which mediates protein-protein interactions with other homologous recombination mediators such as BRCA1, did not significantly affect FANCJ function in response to ICL agents. Finally, while only a minority of FANCJ mutations (∼12%) exhibited LOF in response to ICL, the authors note that the comprehensive mutation library developed here will continue to be useful in characterizing FANCJ mutants and their functionality in response to other stimuli and other types of genomic lesions.Circulating tumor cells (CTC) provide important prognostic information in metastatic castration-resistant prostate cancer (mCRPC), in which the expression of androgen receptor (AR) splice variants is a key prognostic indicator of patient response to enzalutamide and/or abiraterone (enza/abi). However, most patients do not express AR variants, and therefore additional markers are needed. In this study, Gupta et al. leverage CTC exome sequencing and cop. number alteration profiles from men with mCRPC undergoing therapy with enza/abi to identify genomic events associated with therapy resistance. The authors identify a subset of patients who progressed rapidly on enza/abi and bore genomic lesions that were detectable in pooled CTC DNA, including gain of MYCN, BRCA2, and AR, and loss of CHD1, NCOR2, and PTEN. In patients who initially responded and later acquired resistance, CTC sampling uncovered mutations and copy number alterations which converged on genes linked to proliferation, hormone signaling, lineage plasticity and epigenetic signaling, and resistance to cell death. Taken together, these data could form the molecular basis for more effective therapeutic decision making in mCRPC.Tumors commonly down-regulate antigen processing and presentation pathways in order to evade the anti-tumor immune response, which can undermine the efficacy of immune checkpoint blockade. Here, Zhang and colleagues show that Qa-1b—the mouse homolog of the human major histocompatibility complex class I molecule HLA-E—is a key player in how tumors and the immune system interact when antigen presentation is defective. Loss of TAP1 resulted in the establishment of an immunosuppressive tumor microenvironment, but loss of both TAP1 and Qa-1b restored sensitivity to immunotherapy. This effect was mediated at least in part through direct interactions between Qa-1b and natural killer cells, likely through the inhibitory NKG2A receptor through which Qa-1b and HLA-E normally act. Concurrent disruption of TAP1 and Qa-1b reversed immunoresistance by causing increased CD8+ T cell to Treg ratios, increased natural killer cell infiltration and activity, and sensitization to immune checkpoint blockade with anti-PD-1 antibodies. The data suggest that Qa-1b and HLA-E engage in crosstalk with the antigen presentation machinery and represent a key control node that governs the immune response to antigen presentation by tumor cells.

The Evolutionarily Conserved TLDc Domain Defines a New Class of V‐ATPase Interacting Proteins

The kidney-specific V-ATPase regulates physiological acid-base homeostasis, and its dysfunction causes distal renal tubular acidosis (dRTA). We recently found that nuclear receptor coactivator 7 (NCOA7) interacts with the V-ATPase in the kidney, and its deletion in mice resulted in dRTA. NCOA7 belongs to a group of five proteins that contain the evolutionarily conserved TLDc domain, which are known to play a role in the oxidative stress response, and like the V-ATPase, mutations in TLDc proteins lead to serious human neurological diseases. In addition to NCOA7, we found that the TLDc protein, OXR1, interacts with the kidney V-ATPase. Here we asked if the other proteins that make up this family, TBC1D24, TLDC1 and TLDC2, also, interact with the kidney V-ATPase and whether this interaction is mediated by their TLDc domains. Interactions with the V-ATPase were assessed by GST pull-down using mouse kidney lysates and purified GST-tagged TLDc domains of NCOA7, OXR1, TBC1D24, TLDC1 and TLDC2 or the NCOA7 TLDc domain harboring pathogenic mutations (G802A, G815A, S817A, G845A, G896A, L926A, E938A), followed by western blotting. These experiments revealed that the purified TLDc domains of NCOA7, OXR1 and TLDC2, but not TBC1D24 or TLDC1, interact with the kidney-enriched B1 isoform of the V-ATPase. Moreover, the G815A, G845A and G896A mutants found in evolutionarily conserved regions of the NCOA7 TLDc domain did not interact with V-ATPase, L926A and E938A mutations decreased interaction, while S817A or the non-conserved G802A mutation, did not attenuate interaction at all. Since we could not identify an interaction between purified TBC1D24 or TLDC1 and the kidney V-ATPase we investigated the interactions between endogenous TBC1D24, TLDC1 and V-ATPase by co-immunoprecipitation (co-IP) and western blotting. In co-IP studies using mouse kidney lysate, we found that endogenous TBC1D24 interacts with the B1 isoform of the V-ATPase, but not with the more ubiquitous B2 isoform. On the other hand, we did not detect any interaction between endogenous TLDC1 and either the B1 or B2 isoform of the V-ATPase in kidney lysates. Because we were unable to detect TLDC1 in kidney lysates, we performed a co-IP using mouse lung lysate, which expresses high levels of TLDC1. In lung we found that TLDC1 interacts with the B2, but not the B1 subunit of the V-ATPase. However, B1 was expressed at low levels in the lung, which could explain the lack of detectable interaction with TLDC1. Of note, co-IP experiments using equal parts lung and kidney lysate showed a stronger interaction with B2 than B1, suggesting that TLDC1 may preferentially interact with the B2 subunit of the V-ATPase. In the kidney, NCOA7 and OXR1, as well as TBC1D24 and possibly TLDC2 interacted with the V-ATPase and may play a role in V-ATPase-dependent regulation of renal acid-base homeostasis. Furthermore, the evolutionary conserved amino acids within the TLDc domain of Ncoa7 are critical for its interaction with the kidney V-ATPase. Although TLDC1 was not detectable in the kidney, it interacts with the B2 isoform of the V-ATPase in the lung. We conclude, that the TLDc motif is a protein-protein interaction domain that defines a new class of V-ATPase interacting regulatory proteins.

Mutations and Protein Interaction Landscape Reveal Key Cellular Events Perturbed in Upper Motor Neurons with HSP and PLS.

Hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS) are rare motor neuron diseases, which affect mostly the upper motor neurons (UMNs) in patients. The UMNs display early vulnerability and progressive degeneration, while other cortical neurons mostly remain functional. Identification of numerous mutations either directly linked or associated with HSP and PLS begins to reveal the genetic component of UMN diseases. Since each of these mutations are identified on genes that code for a protein, and because cellular functions mostly depend on protein-protein interactions, we hypothesized that the mutations detected in patients and the alterations in protein interaction domains would hold the key to unravel the underlying causes of their vulnerability. In an effort to bring a mechanistic insight, we utilized computational analyses to identify interaction partners of proteins and developed the protein-protein interaction landscape with respect to HSP and PLS. Protein-protein interaction domains, upstream regulators and canonical pathways begin to highlight key cellular events. Here we report that proteins involved in maintaining lipid homeostasis and cytoarchitectural dynamics and their interactions are of great importance for UMN health and stability. Their perturbation may result in neuronal vulnerability, and thus maintaining their balance could offer therapeutic interventions.