Raquel Godoy-Ruiz

Enterovirus D68 (EV-D68) is a plus-strand RNA virus that primarily causes respiratory infections in infants but, in rare cases, has been associated with the pediatric paralytic disease acute flaccid myelitis. We previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that the 3C protease of EV-D68 cleaves the mitochondrial fusion protein Mitofusin 2 near its C-terminal HR2 domain, inducing fragmentation of the mitochondrial network. This, in turn, triggers the formation of mitophagosomes, a hallmark of mitophagy, a selective form of autophagy that recycles mitochondria. Multiple hallmarks of mitophagy are observed during infection, including loss of mitochondrial membrane potential and Parkin translocation to the mitochondria, but mitochondrial degradation is blocked during infection. While autophagy plays multiple roles in enterovirus infection, depleting Mitofusin 2 or transiently overexpressing Mitofusin 2, particularly the cleavage-resistant mutant, specifically reduces EV-D68 release from cells without affecting intracellular titers. Our results show that enteroviruses induce mitophagosomes as vectors for nonlytic release of virions from cells.

Human transglutaminases (hTGs) are Ca²⁺-dependent enzymes that catalyze protein crosslinking, deamidation and other post-translational modifications, thus acting as key stabilizers of tissue architecture and modulators of protein function across diverse physiological contexts. This family comprises eight catalytically active members, TG1-7, the blood coagulation factor FXIII, and the inactive structural protein Band 4.2 of the erythrocyte membrane. Recent structural and biochemical advances have refined our understanding of the molecular principles governing transglutaminase function. Thus, current evidence reveals how domain organization and catalytic architecture integrate calcium binding, nucleotide-dependent regulation in TG2 and proteolytic activation in selected isoforms to control enzymatic activity. In this review, we provide an updated and comprehensive overview of the active hTGs, combining structural, biochemical and functional data to explain how closely related enzymes achieve isoform-specific regulation and distinct biological roles. We further examine how disruption of these mechanisms contributes to human pathology, highlighting representative examples in autoimmunity, inherited disorders and complex diseases. By integrating recent biochemical and structural findings with disease-associated evidence, we aim to offer a coherent framework for understanding how TG regulation underlies their diverse biological functions and clinical relevance.

Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent skin cancer diagnosed worldwide after basal cell carcinoma. CSCC represents a growing global public health challenge due to its higher potential of local invasion, recurrence, and metastasis. Incidence rates of cSCC are projected to increase due to rising exposures to risks factors. Ultraviolet light exposure is the primary cause, and lighter skin pigmentation, immunosuppressive conditions and skin phototype are the primary risk factors. CSCC typically presents as a red, scaly, flat lesion (in situ tumors) or a red, firm, raised lesion with scale or erosion (invasive tumors). Surgical excision remains the standard-of-care for localized cSCC and is often curative. Although, most patients achieve favorable outcomes, a subset of cSCC exhibits a highly aggressive and metastatic phenotype (postoperative recurrence rates are approximately 5%). Addressing the clinical challenge posed by these high-risk cases requires a more comprehensive understanding of the underlying molecular drivers. This review examines the interaction between transglutaminase 2 (TG2) and the G-protein-coupled receptor 56 (GPR56) as a pivotal driver of the aggressive cSCC phenotype. This molecular axis is particularly significant for its role in the maintenance of epidermal cancer stem (ECS) cells, which contribute to tumor progression and therapy resistance. While the definitive link between the TG2-GPR56 complex and systemic metastasis in cSCC is currently being elucidated, significant evidence from analogous malignancies and in vitro keratinocyte models provides a clear mechanistic roadmap for its involvement in tumor invasion.

Clostridioides difficile infection (CDI) is one of the five most urgent bacterial threats in the United States. Furthermore, hypervirulent CDI strains express a third toxin termed the C. difficile binary toxin (CDT), and its molecular mechanism for entering host cells is not fully elucidated. Like other AB-type binary toxins, CDT enters host cells via endosomes. Here we show via surface plasmon resonance and electrochemical impedance spectroscopy that the cell-binding component of CDT, termed CDTb, binds and form pores in lipid bilayers in the absence of its enzymatic component, CDTa. This occurs upon lowering free Ca²⁺ ion concentration, and not by decreasing pH, as found for other binary toxins (i.e., anthrax). Cryogenic electron microscopy (CryoEM), X-ray crystallography, and nuclear magnetic resonance (NMR) studies show that dissociation of Ca²⁺ from a single site in receptor binding domain 1 (RBD1) of CDTb triggers conformational exchange in CDTb. These and structure/function studies of a Ca²⁺-binding double mutant targeting RBD1 (i.e., D623A/D734A) support a model in which dissociation of Ca²⁺ from RBD1 induces dynamic properties in CDTb that enable it to bind and form pores in lipid bilayers.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS‐CoV‐2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross‐priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TN^T, are first generated by grafting an anti‐RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo‐EM data show that the TN^T structure allows simultaneous binding to all six RBD epitopes, demonstrating a high‐avidity neutralizing interaction. Then, by C‐terminal fusion of an anti‐DNGR‐1 scFv to TN^T, the bispecific trimerbody TN^TDNGR‐1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross‐priming. Therapeutic administration of TN^TDNGR‐1, but not TN^T, protects K18‐hACE2 mice from a lethal SARS‐CoV‐2 infection, boosting virus‐specific humoral responses and CD8⁺ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus‐neutralizing antibody activity and demonstrate the therapeutic potential of the Fc‐free strategy that can be used advantageously to provide both immediate and long‐term protection against SARS‐CoV‐2 and other viral infections.

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

Theory and experiments have shown that microsecond folding proteins exhibit characteristic thermodynamic properties that reflect the limited cooperativity of folding over marginal barriers (downhill folding). Those studies have mostly focused on proteins with large α–helical contents and small size, which tend to be the fastest folders. A key open question is whether such properties are also present in the fastest all-beta proteins. We address this issue investigating the unfolding thermodynamics of a collection of WW domains as representatives of the simplest β–sheet fold. WW domains are small microsecond folders, although they do not fold as fast as their α–helical counterparts. In previous work on the NEDD4-WW4 domain, we reported deviations from two-state thermodynamics that were less apparent, and thus suggestive of an incipient downhill scenario. Here we investigate the unfolding thermodynamics of four other WW domains (NEDD4-WW3, YAP65-WW1(L30K), FBP11-WW1 and FBP11-WW2) by performing all of the thermodynamic tests for downhill folding that have been previously developed on α–helical proteins. This set of five WW domains shares low sequence identity and include examples from two specificity classes, thus providing a comprehensive survey. Thermodynamic analysis of the four new WW domains consistently reveals all of the properties of downhill folding equilibria, which are in all cases more marked than what we found before in NEDD4-WW4. Our results show that fast-folding all-beta proteins do share the limited cooperativity and gradual unfolding thermodynamics with fast α-helical proteins, and suggest that the free energy barrier to folding of natural proteins is mostly determined by size and fold topology, and much less by the specific amino-acid sequence.

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein–protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumour formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signalling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signalling, and that drugs designed to target this site may be potent anti-cancer agents.

A window into the cell for vitamin A: Vitamin A is an essential nutrient for mammals, and its metabolites affect diverse biological processes. It is carried in the bloodstream as retinol by retinol binding protein (RBP); a protein called STRA6 is implicated in facilitating retinol translocation across the cell membrane. Chen et al. determined the structure of zebrafish STRA6 to a resolution of 3.9 Å by electron microscopy. A lipophilic cleft is a likely binding site for RBP, and an opening in the cleft may allow retinol to diffuse into the membrane. Unexpectedly, the structure also includes bound calcium-modulated protein, but its function remains unclear. Science , this issue p. 887