Deciphering factors linked with reduced SARS-CoV-2 susceptibility in the Swiss HIV Cohort Study.

BACKGROUND: Factors influencing susceptibility to SARS-CoV-2 remain to be resolved. Using data of the Swiss HIV Cohort Study (SHCS) on 6,270 people with HIV (PWH) and serologic assessment for SARS-CoV-2 and circulating-human-coronavirus (HCoV) antibodies, we investigated the association of HIV-related and general parameters with SARS-CoV-2 infection. METHODS: We analyzed SARS-CoV-2 PCR-tests, COVID-19 related hospitalizations, and deaths reported to the SHCS between January 1, 2020 and December 31, 2021. Antibodies to SARS-CoV-2 and HCoVs were determined in pre-pandemic (2019) and pandemic (2020) bio-banked plasma and compared to HIV-negative individuals. We applied logistic regression, conditional logistic regression, and Bayesian multivariate regression to identify determinants of SARS-CoV-2 infection and Ab responses to SARS-CoV-2 in PWH. RESULTS: No HIV-1-related factors were associated with SARS-CoV-2 acquisition. High pre-pandemic HCoV antibodies were associated with a lower risk of subsequent SARS-CoV-2 infection and with higher SARS-CoV-2 antibody responses upon infection. We observed a robust protective effect of smoking on SARS-CoV-2-infection risk (aOR= 0.46 [0.38,0.56], p=2.6*10-14), which occurred even in previous smokers, and was highest for heavy smokers. CONCLUSIONS: Our findings of two independent protective factors, smoking and HCoV antibodies, both affecting the respiratory environment, underscore the importance of the local immune milieu in regulating susceptibility to SARS-CoV-2.

Multifactorial seroprofiling dissects the contribution of pre-existing human coronaviruses responses to SARS-CoV-2 immunity.

Determination of SARS-CoV-2 antibody responses in the context of pre-existing immunity to circulating human coronavirus (HCoV) is critical for understanding protective immunity. Here we perform a multifactorial analysis of SARS-CoV-2 and HCoV antibody responses in pre-pandemic (N = 825) and SARS-CoV-2-infected donors (N = 389) using a custom-designed multiplex ABCORA assay. ABCORA seroprofiling, when combined with computational modeling, enables accurate definition of SARS-CoV-2 seroconversion and prediction of neutralization activity, and reveals intriguing interrelations with HCoV immunity. Specifically, higher HCoV antibody levels in SARS-CoV-2-negative donors suggest that pre-existing HCoV immunity may provide protection against SARS-CoV-2 acquisition. In those infected, higher HCoV activity is associated with elevated SARS-CoV-2 responses, indicating cross-stimulation. Most importantly, HCoV immunity may impact disease severity, as patients with high HCoV reactivity are less likely to require hospitalization. Collectively, our results suggest that HCoV immunity may promote rapid development of SARS-CoV-2-specific immunity, thereby underscoring the importance of exploring cross-protective responses for comprehensive coronavirus prevention.

The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles.

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-ß to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.

Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1.

Tissue homeostasis is controlled by signaling systems that coordinate cell proliferation, cell growth and cell shape upon changes in the cellular environment. Deregulation of these processes is associated with human cancer and can occur at multiple levels of the underlying signaling systems. To gain an integrated view on signaling modules controlling tissue growth, we analyzed the interaction proteome of the human Hippo pathway, an established growth regulatory signaling system. The resulting high-resolution network model of 480 protein-protein interactions among 270 network components suggests participation of Hippo pathway components in three distinct modules that all converge on the transcriptional co-activator YAP1. One of the modules corresponds to the canonical Hippo kinase cassette whereas the other two both contain Hippo components in complexes with cell polarity proteins. Quantitative proteomic data suggests that complex formation with cell polarity proteins is dynamic and depends on the integrity of cell-cell contacts. Collectively, our systematic analysis greatly enhances our insights into the biochemical landscape underlying human Hippo signaling and emphasizes multifaceted roles of cell polarity complexes in Hippo-mediated tissue growth control.

Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production.

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.

Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome.

Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism.

Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling.

The Hippo (Hpo) pathway is a central determinant of tissue size in both Drosophila and higher organisms. The core of the pathway is a kinase cascade composed of an upstream kinase Hpo (MST1/2 in mammals) and a downstream kinase Warts (Wts, Lats1/2 in mammals), as well as several scaffold proteins, Sav, dRASSF, and Mats. Activation of the core kinase cassette results in phosphorylation and inactivation of the progrowth transcriptional coactivator Yki, leading to increased apoptosis and reduced tissue growth. The mechanisms that prevent inappropriate Hpo activation remain unclear, and in particular, the identity of the phosphatase that antagonizes Hpo is unknown. Using combined proteomic and RNAi screening approaches, we identify the dSTRIPAK PP2A complex as a major regulator of Hpo signaling. dSTRIPAK depletion leads to increased Hpo activatory phosphorylation and repression of Yki target genes in vivo, suggesting this phosphatase complex prevents Hpo activation during development.

The dASPP-dRASSF8 complex regulates cell-cell adhesion during Drosophila retinal morphogenesis.

BACKGROUND: Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-cadherin interactions and are linked to the actin cytoskeleton via the intermediate binding proteins beta-catenin and alpha-catenin. Rather than being static structures, AJs are extensively remodeled during development, allowing the cell rearrangements required for morphogenesis. Several "noncore" AJ components have been identified, which modulate AJs to promote this plasticity but are not absolutely required for cell-cell adhesion. RESULTS: We previously identified dASPP as a positive regulator of dCsk (Drosophila C-terminal Src kinase). Here we show that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. Our genetic and biochemical data suggest that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other's localization. Furthermore, we observed abnormal E-cadherin localization in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or overexpression of Src elicited similar AJ defects. CONCLUSIONS: Because Src is known to regulate AJs in both Drosophila and mammals, we propose that dASPP and dRASSF8 fine tune cell-cell adhesion during development by directing dCsk and Src activity. We show that the dASPP-dRASSF8 interaction is conserved in humans, suggesting that mammalian ASPP1/2 and RASSF8, which are candidate tumor-suppressor genes, restrict the activity of the Src proto-oncogene.

Quantitative interaction proteomics using mass spectrometry.

We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

An integrated workflow for charting the human interaction proteome: insights into the PP2A system.

Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP-MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP-MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP-MS studies on human protein complexes.

PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling.

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of Imd signaling (PIMS), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections.

Early antibodies specific for the neutralizing epitope on the receptor binding subunit of the lymphocytic choriomeningitis virus glycoprotein fail to neutralize the virus.

Lymphocytic choriomeningitis virus (LCMV) is a murine arenavirus whose glycoprotein consists of a transmembrane subunit (GP-2) and a receptor-binding subunit (GP-1). LCMV-neutralizing antibodies (nAbs) are directed against a single site on GP-1 and occur 1 month after the infection of cytotoxic-T-lymphocyte (CTL) deficient mice. In wild-type mice, however, CTLs control early infection, and weak nAb titers emerge very late (after 70 to 150 days) if at all. Production of recombinant GP-1 in native conformation enabled us to study the emergence of GP-1-binding antibodies directed against the neutralizing epitope. By combining binding and neutralization assays, we correlated the development of binding antibodies versus nAbs in wild-type and CTL-deficient mice after infection with different LCMV doses. We found that wild-type mice developed GP-1-specific antibodies already by day 8 after exposure to high but not low doses, demonstrating that naive GP-1-specific B cells were infrequent. Furthermore, the induced antibodies bound to the neutralizing GP-1 epitope but failed to neutralize the virus and therefore were of low affinity. In CTL-deficient mice, where massive viremia quickly levels initial differences in viral load, low and high doses induced low-affinity non-neutralizing GP-1-binding antibodies with kinetics similar to high-dose-infected wild-type mice. Only in CTL-deficient mice, however, the GP-1-specific antibodies developed into nAbs within 1 month. We conclude that LCMV uses a dual strategy to evade nAb responses in wild-type mice. First, LCMV exploits a "hole" in the murine B-cell repertoire, which provides only a small and narrow initial pool of low-affinity GP-1-specific B cells. Second, affinity maturation of the available low-affinity non-neutralizing antibodies is impaired.

Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins.

The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal alpha-helix. An alpha-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second alpha-helical peptide corresponding to a predicted C-terminal alpha-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of approximately 230 and approximately 440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.