Biology subjects

Dominguez, F.

Publications and source records attributed to Dominguez, F..

9 recordsLinked to original sources

bioRxiv2025

Mucosal delivery of influenza antigens using a replication deficient adenovirus supports broadly reactive antibody responses and heterologous viral immunity in the respiratory tract of animals

Systemically administered influenza vaccines provide strain-limited protection, while influenza infection of the respiratory epithelium supports development of lung resident memory B and T cells and more broadly reactive antibody responses. To test whether local antigen delivery is critical for establishing broad immunity, we directly compared respiratory tract and systemic delivery of influenza antigens to mice and hamsters using a replication-deficient adenovirus serotype 5 vector (Ad5[E1-,E2b-,E3-]). Both immunization routes elicited antibody responses in the lower respiratory tract and antigen-specific B and T cells in the draining lymph node. However, only intranasal immunization established lung-resident memory B and T cells, induced IgA responses in the upper respiratory tract directly at the site of viral entry and supported generation of IgA and IgG antibodies that bound antigenically drifted and distantly related influenza strains, including those of avian origin. Intranasal immunization accelerated viral clearance following heterologous virus challenge and was associated with limited pulmonary inflammation and fibrosis. Thus, intranasal immunization with the immunologically stealthy Ad5[E1-,E2b-,E3-] platform supports respiratory and systemic immunity to divergent influenza strains in the absence of overt lung immunopathology, suggesting that local antigen delivery may be key to development of more broadly protective "universal" flu vaccines.

immunology↗

bioRxiv2023

Alphavirus-induced transcriptional and translational shutoffs play major roles in blocking the formation of stress granules

Alphavirus infections cause multiple alterations in the intracellular environment that can have both positive and negative effects on viral replication. The Old World alphaviruses, such as Sindbis (SINV), chikungunya (CHIKV), and Semliki Forest viruses, hinder the ability of vertebrate cells to form stress granules (SGs). Previously, this inhibitory function was attributed to the hypervariable domain (HVD) of nsP3, which sequesters the key components of SGs, G3BP1 and G3BP2, and to the nsP3 macro domain. The macro domain possesses ADP-ribosylhydrolase activity, which can diminish the ADP-ribosylation of G3BP1 during viral replication. However, our recent findings do not support the prevailing notions. We demonstrate that the interactions between SINV- or CHIKV-specific nsP3s and G3BPs, and the ADP-ribosylhydrolase activity are not major contributors to the inhibitory process, at least when nsP3 is expressed at biologically relevant levels. Instead, the primary factors responsible for suppressing SG formation are virus-induced transcriptional and translational shutoffs that rapidly develop within the first few hours post infection. Poorly replicating SINV variants carrying mutated nsP3 HVD still inhibit SG development even in the presence of NaAs. Conversely, SINV mutants lacking transcription and/or translation inhibitory functions lose their ability to inhibit SGs, despite expressing high levels of wt nsP3. Moreover, we found that stable cell lines expressing GFP-nsP3 fusions retain the capacity to form SGs when exposed to sodium arsenite. However, our results do not rule out a possibility that additional virus-induced changes in cell biology may contribute to the suppression of SG formation. ImportanceOur study highlights the mechanisms behind the cells resistance to SG formation after infection with Old World alphaviruses. Shortly after infection, the replication of these viruses hinders the cells ability to form SGs, even when exposed to chemical inducers such as sodium arsenite. This resistance is primarily attributed to virus-induced transcriptional and translational shutoffs, rather than interactions between the viral nsP3 and the key components of SGs, G3BP1/2, or the ADP-ribosylhydrolase activity of nsP3 macro domain. While interactions between G3BP and nsP3 are essential for the formation of viral replication complexes, their role in regulating SG development appears to be minimal, if any. Cells harboring replicating virus-specific RNA with modified abilities to inhibit transcription and/or translation, but encoding wt nsP3, retain the capacity for SG development. Understanding these mechanisms of regulation of SG development contributes to our knowledge of viral replication and the intricate relationships between alphaviruses and host cells.

microbiology↗

bioRxiv2023

Amino acid substitutions hydrophilizing the core of titin domains cause dilated cardiomyopathy

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the increase in hydrophilicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophilizing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

biochemistry↗

bioRxiv2021

Site-specific recognition of SARS-CoV-2 nsp1 protein with a tailored titanium dioxide nanoparticle

The ongoing world-wide Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic shows the need for new sensing and therapeutic means against the CoV viruses. The SARS-CoV-2 nsp1 protein is important, both for replication and pathogenesis, making it an attractive target for intervention. In recent years nanoparticles have been shown to interact with peptides, ranging in size from single amino acids up to proteins. These nanoparticles can be tailor-made with specific functions and properties including bioavailability. To the best of our knowledge, in this study we show for the first time that a tailored titanium oxide nanoparticle interacts specifically with a unique site of the full-length SARS-CoV-2 nsp1 protein. This can be developed potentially into a tool for selective control of viral protein functions.

molecular biology↗

bioRxiv2021

NAP1L1 and NAP1L4 binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein is bivalent and requires phosphorylation

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last two decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins, but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication; ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD; iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD; iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD and HVD phosphorylation is mediated by CK2 kinase; v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCECellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We showed that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

microbiology↗

bioRxiv2021

1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution.

Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the C{beta} of the apo form of the full-lengthSARS-CoV-2 nsp1 including folded domain together with the flaking N- and C-terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.

molecular biology↗

bioRxiv2020

Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein

Decades of insufficient control resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe and millions already suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a pre-requisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has detectable stimulatory effect(s) on CHIKV replication, and the Fhl1 KO cell lines demonstrate slower infection spread. The NMR-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps with that of another pro-viral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive positive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP- binding sites remain viable and develop spreading infection in multiple cell types. Thus, such modifications of HVD may improve live CHIKV vaccine candidates in terms of their safety and stability of the attenuated phenotype. IMPORTANCEReplication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 protein contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive pro-viral functions. Elimination of FHL1-binding site in nsP3 HVD can be used for the development of stable, live attenuated vaccine candidates.

microbiology↗

bioRxiv2020

Nanomechanical phenotypes in cMyBP-C mutants that cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C mutant domains. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN, and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, non-pathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

biophysics↗

bioRxiv2020

Conserved cysteines in titin sustain the mechanical function of cardiomyocytes

The protein titin determines cardiomyocyte contraction and truncating variants in the titin gene (TTN) are the most common cause of dilated cardiomyopathy (DCM). Different to truncations, missense variants in TTN are currently classified as variants of uncertain significance due to their high frequency in the population and the absence of functional annotation. Here, we report the regulatory role of conserved, mechanically active titin cysteines, which, contrary to current views, we uncover to be reversibly oxidized in basal conditions leading to isoform- and force-dependent modulation of titin stiffness and dynamics. Building on our functional studies, we demonstrate that missense mutations targeting a conserved titin cysteine alter myocyte contractile function and cause DCM in humans. Our findings have a direct impact on genetic counselling in clinical practice. One sentence summaryMutations targeting cysteines key to the mechanoredox control of titin cause human dilated cardiomyopathy

physiology↗