Read the full paper: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

Azo dyes are widespread in the environment, and humans ingest a range of these dyes present in food and drugs. The human gut microbiome significantly impacts the fate of ingested azo dyes primarily th...

AbstractApolipoprotein E4 (ApoE4), one of the three common isoforms of ApoE, differs from ApoE3 and ApoE2 by just one amino acid substitution each. Yet these changes in ApoE4 profoundly impact protein structure and function to make it the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). ApoE4 exhibits a tendency to form oligomers (e.g., tetramers) that complicate high-resolution structural characterization in aqueous environments. Very little is known about the self-associated oligomeric structures. In this study, we employed ultracentrifugation, native mass spectrometry (MS), and fast photochemical oxidation of proteins (FPOP), a mass spectrometry-based protein footprinting approach, to investigate conformational differences among wild-type ApoE4 and two of its mutant variants ApoE4(V236E) (Jacksonville, JV) and ApoE4(V236E/L268E) (VL). These substitutions shift the oligomeric equilibrium toward dimeric and monomeric states, respectively, and allow the tetramer state to be compared with the dimer and monomer. Comparative footprinting reveals that conformational changes driven by these mutations result in altered solvent accessibility in key regions of the protein, suggesting disruption of interfaces responsible for oligomerization and altered access of lipid-binding regions. Given ApoE4’s central role in lipid metabolism and its altered interaction with lipid surfaces compared to other isoforms, these structural changes likely modulate lipid binding affinity and may contribute to its pathological role in AD. Our findings highlight the importance of conformational states in ApoE4 function and provide new insights into the structural basis of its disease-associated behavior.

AbstractRecent studies showed that several widely used glucagon-like peptide-1 receptor agonists (GLP-1RAs) can self-assemble into dynamic oligomeric species and ultimately less-regular, non-amyloid aggregates. While mature aggregates can be characterized by high-resolution microscopy methods, probing the conformational dynamics of early-stage oligomeric species remains challenging, which limits our understanding of how the misfolding and aggregation propensity of GLP-1RAs is modulated by physiologically relevant conditions such as protein concentration or the presence of metal ions. In this work, multiple structural mass spectrometry (MS) methods were used in combination to resolve the earliest molecular changes that lead to semaglutide oligomerization. These structural MS methods together reveal important information such as the heterogeneity and stoichiometry of oligomers, their structural/thermal stability, protein conformational changes, the molecular interface for self-assembly, relative monomer orientation in oligomers, and aggregation pathway of the resulting oligomeric species, both with and without metal ions present. We found that semaglutide forms oligomers up to 10-mers within 24 h; among seven essential metals for humans, low micromolar concentrations of Cu2+ and Zn2+ ions drive the formation of higher-order and more abundant oligomeric species, an effect attributed to metal binding-mediated stabilization of small oligomers. Oligomeric species are associated via hydrophobic C-termini in a parallel orientation. N-termini and the lipid chain are, instead, loosely engaged and available for high-affinity binding to human serum albumin (HSA), an interaction that facilitates the dissociation of oligomeric species and is further enhanced when HSA is glycated. Our findings underscore the power of structural MS to track early-stage oligomerization.

Chen and colleagues identify CLCC1 as the functional substrate downstream of torsin AAA+ ATPases. TorsinA depletion disrupts CLCC1’s oligomeric state and triggers massive lipid deposition within the ER. Importantly, ectopic expression of CLCC1 overcomes torsinA loss and fully reverses the lipid mis-deposition.

A crucial aspect of inhibitory synaptic transmission is the ability of gephyrin to build a scaffold by multimerization. To test whether the oligomeric state of gephyrin is dependent on cysteines and if those are redox-sensitive, we tested whether gephyrin presents redox-sensitive behavior in native tissue. Therefore, we perfused mice with N-ethyl-maleimide (NEM)-containing PBS to stabilize free thiols and avoid unspecific oxidation. In subsequent Western blot analysis, we either used the reductant dithiothreitol (DTT) or omitted it, allowing to compare the presence of redox-dependent gephyrin species (Fig. 1A). In brain lysates, the absence of a reductant resulted in bands corresponding to a molecular weight of approximately 300 kDa representing multimeric gephyrin species, whereas reduction of gephyrin led exclusively to monomeric gephyrin (100 kDa, Fig. 1A). The fractional abundance of multimeric gephyrin was approximately 20% of total gephyrin. Based on this finding, we hypothesized that gephyrin cysteines are redox-sensitive and that thiol oxidation modulates intermolecular interactions, thereby influencing gephyrin multimerization, which is a crucial parameter for its synaptic function35. Thus, we wondered which surface cysteines of gephyrin could be important for multimerization. Based on crystal structures of G- and E-domain36,37, the identification of palmitoylation26, as well as the disordered nature of the...

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DegP is a periplasmic protease-chaperone essential for protein quality control and virulence factor trafficking in Gram-negative bacteria. In its apo form, DegP adopts a dynamic ensemble of oligomers derived from trimer building blocks through two competing self-assembly pathways. Upon engaging client proteins, apo DegP oligomers redistribute into discrete cage structures inside which the clients are encapsulated. The cage ensembles formed depend on the size of the bound clients, and notably can include 12mers, 24mers, and 60mers. Previous studies mapped the DegP oligmeric landscape using dynamic light scattering, analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and electron cryomicroscopy, modalities which in general report ensemble averages and often cannot directly delineate closely related coexisting species. Here, we apply charge detection mass spectrometry (CDMS) to directly measure the masses of individual DegP ions, resolving the complete oligomeric distribution in the absence and presence of four clients of increasing size. We reveal previously undetected odd-numbered oligomers and quantify the relative abundance of each assembly. Through heat-cool cycling CDMS experiments, we track cage distribution changes and reveal client protection and refolding, providing a direct view of the chaperone capabilities of DegP. These results further establish CDMS as a powerful single-molecule tool for dissecting heterogeneous protein assembly landscapes.

Nitrogen-fixing Nostoc species form symbiotic relationships with diverse plants, yet the role of specialized metabolites in these interactions remains poorly understood. Here, we identify a previously...