The MIA40 relay system mediates the import of small cysteine-rich proteins into the intermembrane mitochondrial space (IMS). MIA40 substrates are synthesized in the cytosol and assumed to be disordered in their reduced state in this compartment. As they cross the outer mitochondrial membrane, MIA40 promotes the oxidation of critical native disulfides to facilitate folding, trapping functional species in the IMS. Here, we study the redox-controled folding of TRIAP1, a small cysteine-rich protein with moonlighting function: regulating phospholipid trafficking between mitochondrial membranes in the IMS and preventing apoptosis in the cytosol. TRIAP1 dysregulation is connected to oncogenesis. Although TRIAP1 contains a canonical twin CX9C motif, its sequence characteristics and folding pathway deviate from typical MIA40 substrates. In its reduced state, TRIAP1 rapidly populates a hydrophobic collapsed, alpha-helical, and marginally stable molten globule. This intermediate biases oxidative folding towards a non-native Cys37-Cys47 kinetic trap, slowing the reaction. MIA40 accelerates TRIAP1 folding rate by 30-fold, bypassing the formation of this folding trap. MIA40 drives the oxidation of the inner disulfide bond Cys18-Cys37, and subsequently, it can catalyze the formation of the outer disulfide bond Cys8-Cys47 to attain the native two-disulfide-bridged structure. We demonstrate that, unlike most MIA40 substrates, TRIAP1's folding pathway is strongly constrained by the structural requirements for its function in phospholipid traffic at the IMS. The obligatory population of a reduced, alpha-helical, metastable molten globule in the cytoplasm may explain TRIAP1's connection to the p53-dependent cell survival pathway, constituting a remarkable example of a functional molten globule state.

The mammarenavirus Junín (JUNV) is the causative agent of Argentine hemorrhagic fever, a severe disease of public health concern. The most abundant viral protein is the nucleoprotein (NP), a multifunctional, two-domain protein with the primary role as structural component of the viral nucleocapsids, used as template for viral polymerase RNA synthesis activities. Here, we report that the C-terminal domain (CTD) of the attenuated Candid#1 strain of the JUNV NP can be purified as a stable soluble form with a secondary structure in line with known NP structures from other mammarenaviruses. We show that the JUNV NP CTD interacts with the viral matrix protein Z in vitro, and that the full-length NP and Z interact with each other in cellulo, suggesting that the NP CTD is responsible for this interaction. This domain comprises an arrangement of four acidic residues and a histidine residue conserved in the active site of exoribonucleases belonging to the DEDDh family. We show that the JUNV NP CTD displays metal-ion-dependent nuclease activity against DNA and single- and double-stranded RNA, and that this activity is impaired by the mutation of a catalytic residue within the DEDDh motif. These results further support this activity, not previously observed in the JUNV NP, which could impact the mechanism of the cellular immune response modulation of this important pathogen.

Background: Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and high-risk adults, in part by subverting host immunity. The RSV G glycoprotein’s central conserved cysteine rich domain (CCD) contains a CX3C motif implicated in immune modulation, but the relationship between its redox state, structural conformation, and immune modulatory function remains unclear. Methods: In this study, we recombinantly expressed a CCD-derived peptide (Gpep, residues 149–196), determined its redox-dependent folding by reversed-phase HPLC (RP-HPLC) and biophysical analyses, and assessed its function using murine dendritic cell and human neutrophil assays alongside pediatric serology. Results: Kinetic analyses by RP‑HPLC and biophysical techniques showed that reduced Gpep rapidly folds through a predominant intermediate to yield an oxidized monomer; conversely, higher concentrations drive intermolecular disulfide isomerization and covalent oligomer formation. Functionally, Gpep inhibited dendritic cell activation elicited by both LPS- and UV-inactivated RSV. In addition, Gpep suppressed multiple human neutrophil responses, including chemotaxis, CD11b upregulation, reactive oxygen species production, myeloperoxidase release, and NET formation, without inducing cytotoxicity. In contrast, oligomerized Gpep lacked immunosuppressive activity. Serological analysis of an ambulatory pediatric cohort (0–72 months) showed a transient increase in the anti-F/anti-G IgG ratio following early RSV exposures, consistent with preferential maturation of F-directed responses. Discussion: We propose a redox‑dependent immune‑evasion model in which secreted, monomeric G mediates transient immunosuppression that is halted by disulfide‑driven oligomerization of membrane‑bound G and F proteins. These findings support a mechanistic association between the redox state of RSV G, its oligomeric behavior, and its immunomodulatory properties.