Alpha-synucleinopathies, characterized by extracellular alpha-synuclein (aSyn) accumulation and aggregation, have been linked to neurological disorders including Parkinson disease (PD) and multiple system atrophy (MSA). aSyn interacts with membrane proteins, phospholipids, and cholesterol, leading to membrane damage and inflammation. This interaction heightens the likelihood of aSyn engaging with P2RX7, a non-selective cationic transmembrane receptor that is primarily overexpressed in immune and neural cells. Activation of P2RX7 by aSyn is implicated in neuronal degeneration, potentially causing pore dilation and increased inflammation. However, the precise molecular mechanisms and binding sites for this interaction, as well as the conformational dynamics of P2RX7 in response to aSyn, remain unclear. We attempted to elucidate the molecular mechanisms and binding sites for P2RX7-aSyn interaction, by integrating the data curation, molecular docking, and molecular dynamics (MD) simulations coupled with structural analyses. We elucidated interactions between P2RX7 and the N-terminal domain (NTD) of aSyn using cryo-EM structures of P2RX7 in both ATP-bound and unbound states and assessed how aSyn influences P2RX7 structural and functional dynamics. Initially, the analyses revealed that aSyn interactomes are mainly involved in regulating mitochondrial homeostasis, while P2RX7 interactors are linked to receptor internalization and calcium transport. Molecular docking with six tools identified that aSyn-NTD fragments preferentially bind to the proximal region of P2RX7 transmembrane domain. Microsecond all atom MD simulations in a POPS lipid bilayer showed significant atomic fluctuations, particularly in the head region, lower body, and large loop of P2RX7 cytoplasmic domain. Secondary structure analysis indicated unfolding in regions related to pore dilation and receptor desensitization. Further by contact-based and solvent accessibility analyses, along with protein structure network (PSN) studies, we identified crucial residues involved in P2RX7-aSyn interactions. This insight deepens our understanding of how aSyn and P2RX7 interact, offering a detailed atomic view of the structural and functional changes that occur during these interactions. This understanding could advance our grasp of neurodegenerative diseases and be vital for devising future preventive and therapeutic strategies. ### Competing Interest Statement The authors have declared no competing interest.

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing–remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

Autophagy is a key clearance process to recycle damaged cellular components. One important upstream regulator of autophagy is ULK1 kinase. Several three-dimensional structures of the ULK1 catalytic domain are available, but a comprehensive study, including molecular dynamics, is missing. Also, an exhaustive description of ULK1 alterations found in cancer samples is presently lacking. We here applied a framework which links -omics data to structural protein ensembles to study ULK1 alterations from genomics data available for more than 30 cancer types. We predicted the effects of mutations on ULK1 function and structural stability, accounting for protein dynamics, and the different layers of changes that a mutation can induce in a protein at the functional and structural level. ULK1 is down-regulated in gynecological tumors. In other cancer types, ULK2 could compensate for ULK1 downregulation and, in the majority of the cases, no marked changes in expression have been found. 36 missense mutations of ULK1, not limited to the catalytic domain, are co-occurring with mutations in a large number of ULK1 interactors or substrates, suggesting a pronounced effect of the upstream steps of autophagy in many cancer types. Moreover, our results pinpoint that more than 50% of the mutations in the kinase domain of ULK1, here investigated, are predicted to affect protein stability. Three mutations (S184F, D102N, and A28V) are predicted with only impact on kinase activity, either modifying the functional dynamics or the capability to exert effects from distal sites to the functional and catalytic regions. The framework here applied could be extended to other protein targets to aid the classification of missense mutations from cancer genomics studies, as well as to prioritize variants for experimental validation, or to select the appropriate biological readouts for experiments.

Autophagy is a conserved and essential intracellular mechanism for the removal of damaged components. Since autophagy deregulation is linked to different kinds of pathologies, it is fundamental to gain knowledge on the fine molecular and structural details related to the core proteins of the autophagy machinery. Among these, the family of human ATG8 proteins plays a central role in recruiting other proteins to the different membrane structures involved in the autophagic pathway. Several experimental structures are available for the members of the ATG8 family alone or in complex with their different biological partners, including disordered regions of proteins containing a short linear motif called LC3 interacting motif. Recently, the first structural details of the interaction of ATG8 proteins with biological membranes came into light. The availability of structural data for human ATG8 proteins has been paving the way for studies on their structure-function-dynamic relationship using biomolecular simulations. Experimental and computational structural biology can help to address several outstanding questions on the mechanism of human ATG8 proteins, including their specificity toward different interactors, their association with membranes, the heterogeneity of their conformational ensemble, and their regulation by post-translational modifications. We here summarize the main results collected so far and discuss the future perspectives within the field and the knowledge gaps. Our review can serve as a roadmap for future structural and dynamics studies of the ATG8 family members in health and disease.

Macroautophagy/autophagy is a cellular process to recycle damaged cellular components, and its modulation can be exploited for disease treatments. A key autophagy player is the ubiquitin-like protein MAP1LC3B/LC3B. Mutations and changes in MAP1LC3B expression occur in cancer samples. However, the investigation of the effects of these mutations on MAP1LC3B protein structure is still missing. Despite many LC3B structures that have been solved, a comprehensive study, including dynamics, has not yet been undertaken. To address this knowledge gap, we assessed nine physical models for biomolecular simulations for their capabilities to describe the structural ensemble of MAP1LC3B. With the resulting MAP1LC3B structural ensembles, we characterized the impact of 26 missense mutations from pan-cancer studies with different approaches, and we experimentally validated our prediction for six variants using cellular assays. Our findings shed light on damaging or neutral mutations in MAP1LC3B, providing an atlas of its modifications in cancer. In particular, P32Q mutation was found detrimental for protein stability with a propensity to aggregation. In a broader context, our framework can be applied to assess the pathogenicity of protein mutations or to prioritize variants for experimental studies, allowing to comprehensively account for different aspects that mutational events alter in terms of protein structure and function. Abbreviations: ATG: autophagy-related; Cα: alpha carbon; CG: coarse-grained; CHARMM: Chemistry at Harvard macromolecular mechanics; CONAN: contact analysis; FUNDC1: FUN14 domain containing 1; FYCO1: FYVE and coiled-coil domain containing 1; GABARAP: GABA type A receptor-associated protein; GROMACS: Groningen machine for chemical simulations; HP: hydrophobic pocket; LIR: LC3 interacting region; MAP1LC3B/LC3B microtubule associated protein 1 light chain 3 B; MD: molecular dynamics; OPTN: optineurin; OSF: open software foundation; PE: phosphatidylethanolamine, PLEKHM1: pleckstrin homology domain-containing family M 1; PSN: protein structure network; PTM: post-translational modification; SA: structural alphabet; SLiM: short linear motif; SQSTM1/p62: sequestosome 1; WT: wild-type.

By assisting in the proteolysis, disaggregation and refolding of the aggregated proteins, Caseinolytic proteases (Clps) enhance the cellular survival under stress conditions. In the current study, comparative roles of two such Clps, ClpA (involved in proteolysis) and ClpB (involved in protein disaggregation and refolding) in the survival of Salmonella Typhimurium (S. Typhimurium) under different stresses and in virulence have been investigated. clpA and clpB gene deletion mutant strains (∆clpA and ∆clpB) of S. Typhimurium have been hypersensitive to 42 °C, HOCl and paraquat. However, the ∆clpB strain was comparatively much more susceptible (p < 0.001) to the above stresses than ∆clpA strain. ∆clpB strain also showed reduced survival (p < 0.001) in poultry macrophages. The hypersusceptibilities of ∆clpB strain to oxidants and macrophages were restored in plasmid based complemented (∆clpB + pclpB) strain. Further, the ∆clpB strain was defective for colonization in the poultry caecum and showed decreased dissemination to the spleen and liver. Our findings suggest that the role of ClpB is more important than the role of ClpA for the survival of S. Typhimurium under stress and colonization in chickens.

BACKGROUND:Cholesterol in lipid raft plays crucial role on cancer cell survival during metastasis of cancer cells. Cancer cells are reported to enrich cholesterol in lipid raft which make them more susceptible to cell death after cholesterol depletion than normal cells. Methyl-β-cyclodextrin (MβCD), an amphipathic polysaccharide known to deplete the membrane cholesterol, induces cell death selectively in cancer cells. Present work was designed to identify the major form of programmed cell death in membrane cholesterol depleted cancer cells (MDA-MB 231 and 4T1) and its impact on migration efficiency of cancer cells.METHODS:Membrane cholesterol alteration and morphological changes in 4T1 and MDA-MB 231 cancer cells by MβCD were measured by fluorescent microscopy. Cell death and cell proliferation were observed by PI, AO/EB and MTT assay respectively. Programme cell death was confirmed by flow cytometer. Caspase activation was assessed by MTT and PI after treatments with Z-VAD [OME]-FMK, mitomycin c and cycloheximide. Necroptosis, autophagy, pyroptosis and paraptosis were examined by cell proliferation assay and flow cytometry. Relative quantitation of mRNA of caspase-8, necroptosis and autophagy genes were performed. Migration efficiency of cancer cells were determined by wound healing assay.RESULTS:We found caspase independent cell death in cholesterol depleted MDA-MB 231 cells which was reduced by (3-MA) an autophagy inhibitor. Membrane cholesterol depletion neither induces necroptosis, paraptosis nor pyroptosis in MDA-MB 231 cells. Subsequent activation of caspase-8 after co-incubation of mitomycin c and cycloheximide separately, restored the cell viability in cholesterol depleted MDA-MB 231 cells. Down regulation of caspase-8 mRNA in cholesterol depleted cancer cells ensures that caspase-8 indirectly promotes the induction of autophagy. In another experiment we have demonstrated that membrane cholesterol depletion reduces the migration efficiency in cancer cells.CONCLUSION:Together our experimental data suggests that membrane cholesterol is the crucial for the recruitment and activation of caspase-8 as well as its non-apoptotic functions in cancer cells. Enriched cholesterol in lipid raft of cancer cells may be regulating the cross talk between caspase-8 and autophagy machineries to promote their survival and migration. Therefore it can be explored to understand and address the issues of chemotherapeutic and drugs resistance.

Matrix metalloproteinase-11 (MMP-11) is known to be highly expressed in metastatic and most invasive forms of tumors. Being selectively expressed in tumor tissues, MMP-11 is a promising target for immunotherapy against tumors. Here, we report the production of a thioredoxin-tagged bioactive recombinant chicken MMP-11 (cMMP-11) peptide excluding the secretory signal and propeptide in Escherichia coli T7 Express lysY using pET32b(+) vector. High-level expression and purification of the bioactive peptide were achieved by induction with 1.0 mM isopropyl-β-d-thiogalactopyranoside for 4 H at 37 °C followed by affinity chromatography under denaturing condition and slow dialysis. The recombinant peptide exhibited both caseinolytic and gelatinase activities without requiring activation by 4-aminophenylmercuric acetate. The antisera raised against the peptide in rabbits showed a strong reaction with the whole recombinant peptide as well as 37 kDa cMMP-11 mature peptide and cross-reactivity with a 43 kDa protein in murine breast tumor of 4T1 origin in Western blot analysis. The 43 kDa protein in the tumor homogenate showed immunoreactivity with a monoclonal antibody against human MMP-11, suggesting it to be murine MMP-11 having cross-reactivity with the antisera raised against cMMP-11 peptide. Altogether, the study characterized the production of a bioactive and immunogenic recombinant cMMP-11 peptide in E. coli.