HIV-1 Nef Changes the Proteome of T Cells Extracellular Vesicles Depleting IFITMs and Other Antiviral Factors.

Extracellular vesicles (EVs) are biomolecule carriers for intercellular communication in health and disease. Nef is a HIV virulence factor that is released from cells within EVs and is present in plasma EVs of HIV-1 infected individuals. We performed a quantitative proteomic analysis to fully characterize the Nef-induced changes in protein composition of T cell-derived EVs and identify novel host targets of HIV. Several proteins with well-described roles in infection or not previously associated with HIV pathogenesis were specifically modulated by Nef in EVs. Among the downregulated proteins are the interferon-induced transmembrane 1, 2, and 3 (IFITM1-3) proteins, broad-spectrum antiviral factors known to be cell-to-cell transferable by EVs. We demonstrate that Nef depletes IFITM1-3 from EVs by excluding these proteins from the plasma membrane and lipid rafts, which are sites of EVs biogenesis in T cells. Our data establish Nef as a modulator of EVs' global protein content and as an HIV factor that antagonizes IFITMs.

New Scaffolds of Proteasome Inhibitors: Boosting Anticancer Potential by Exploiting the Synergy of In Silico and In Vitro Methodologies.

Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin-proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.

Two Functional Variants of AP-1 Complexes Composed of either γ2 or γ1 Subunits Are Independently Required for Major Histocompatibility Complex Class I Downregulation by HIV-1 Nef.

The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation.IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.

Glutathione in multiple sclerosis: more than just an antioxidant?

Oxidative stress has been strongly implicated in both the inflammatory and neurodegenerative pathological mechanisms in multiple sclerosis (MS). In response to oxidative stress, cells increase and activate their cellular antioxidant mechanisms. Glutathione (GSH) is the major antioxidant in the brain, and as such plays a pivotal role in the detoxification of reactive oxidants. Previous research has shown that GSH homeostasis is altered in MS. In this review, we provide a comprehensive overview on GSH metabolism in brain cells, with a focus on its involvement in MS. The potential of GSH as an in vivo biomarker in MS is discussed, along with a short overview of improvements in imaging methods that allow non-invasive quantification of GSH in the brain. These methods might be instrumental in providing real-time measures of GSH, allowing the assessment of the oxidative state in MS patients and the monitoring of disease progression. Finally, the therapeutic potential of GSH in MS is discussed.