Title: β2-microglobulin triggers NLRP3 inflammasome activation in tumor-associated macrophages to promote multiple myeloma progression.

Protein aggregation into amyloid fibrils is the archetype of aberrant biomolecular self-assembly processes, with more than 50 associated diseases that are mostly uncurable. Understanding aggregation mechanisms is thus of fundamental importance and goes in parallel with the structural characterization of the transient oligomers formed during the process. Oligomers have been proven elusive to high-resolution structural techniques, while the large sizes and long time scales, typical of aggregation processes, have limited the use of computational methods to date. To surmount these limitations, we here present multi- e GO, an atomistic, hybrid structure-based model which, leveraging the knowledge of monomers conformational dynamics and of fibril structures, efficiently captures the essential structural and kinetics aspects of protein aggregation. Multi- e GO molecular dynamics simulations can describe the aggregation kinetics of thousands of monomers. The concentration dependence of the simulated kinetics, as well as the structural features of the resulting fibrils, are in qualitative agreement with in vitro experiments carried out on an amyloidogenic peptide from Transthyretin, a protein responsible for one of the most common cardiac amyloidoses. Multi- e GO simulations allow the formation of primary nuclei in a sea of transient lower-order oligomers to be observed over time and at atomic resolution, following their growth and the subsequent secondary nucleation events, until the maturation of multiple fibrils is achieved. Multi- e GO, combined with the many experimental techniques deployed to study protein aggregation, can provide the structural basis needed to advance the design of molecules targeting amyloidogenic diseases.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic example is human beta‐2 microglobulin (β2m), which, when accumulated in patients, gives rise to dialysis‐related amyloidosis. Interestingly, although the physiologic concentration of β2m in mice is five times higher than that found in human patients, no amyloid deposits are observed in mice. Moreover, murine β2m (mβ2m) not only displays a lower amyloid propensity both in vivo and in vitro but also inhibits the aggregation of human β2m in vitro. Here, we compared human and mβ2m for their aggregation propensity, ability to form soluble oligomers, stability, three‐dimensional structure and dynamics. Our results indicate that mβ2m low‐aggregation propensity is due to two concomitant aspects: the low‐aggregation propensity of its primary sequence combined with the absence of high‐energy amyloid‐competent conformations under native conditions. The identification of the specific properties determining the low‐aggregation propensity of mouse β2m will help delineate the molecular risk factors which cause a folded protein to aggregate.

Beta-2 microglobulin (β2m) is a protein responsible for a pathologic condition, known as dialysis-related amyloidosis (DRA), caused by its aggregation and subsequent amyloid formation. A naturally occurring mutation of β2m, D76N, presents a higher amyloidogenic propensity compared to the wild type counterpart. Since the three-dimensional structure of the protein is essentially unaffected by the mutation, the increased aggregation propensity of D76N has been generally ascribed to its lower thermodynamic stability and increased dynamics. In this study we compare the equilibrium unfolding and the aggregation propensity of wild type β2m and D76N variant at different experimental conditions. Our data revealed a surprising effect of the D76N mutation in the residual structure of the denatured state, which appears less compact than that of the wild type protein. A careful investigation of the structural malleability of the denatured state of wild type β2m and D76N pinpoint a clear role of the denatured state in triggering the amyloidogenic propensity of the protein. The experimental results are discussed in the light of the previous work on β2m and its role in disease.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a severe and lethal neurodegenerative disease. Upon specific point mutations in the SERPINI1gene-coding for the human protein neuroserpin (NS) the resulting pathologic NS variants polymerize and accumulate within the endoplasmic reticulum of neurons in the central nervous system. To date, embelin (EMB) is the only known inhibitor of NS polymerization in vitro. This molecule is capable of preventing NS polymerization and dissolving preformed polymers. Here, we show that lowering EMB concentration results in increasing size of NS oligomers in vitro. Moreover, we observe that in cells expressing NS, the polymerization of G392E NS is reduced, but this effect is mediated by an increased proteasomal degradation rather than polymerization impairment. For these reasons we designed a systematic chemical evolution of the EMB scaffold aimed to improve its anti-polymerization properties. The effect of EMB analogs against NS polymerization was assessed in vitro. None of the EMB analogs displayed an anti-polymerization activity better than the one reported for EMB, indicating that the EMB–NS interaction surface is very specific and highly optimized. Thus, our results indicate that EMB is, to date, still the best candidate for developing a treatment against NS polymerization.

Presentation of pathogen-derived epitopes by major histocompatibility complex I (MHC-I) can lead to the activation and expansion of specific CD8 + T cell clones, eventually resulting in the destruction of infected target cells. Altered peptide ligands (APLs), designed to elicit immunogenicity toward a wild-type peptide, may affect the overall stability of MHC-I/peptide (pMHC) complexes and modulate the recognition by T cell receptors (TCR). Previous works have demonstrated that proline substitution at position 3 (p3P) of different MHC-restricted epitopes, including the immunodominant LCMV-derived epitope gp33 and escape variants, may be an effective design strategy to increase epitope immunogenicity. These studies hypothesized that the p3P substitution increases peptide rigidity, facilitating TCR binding. Here, molecular dynamics simulations indicate that the p3P modification rigidifies the APLs in solution predisposing them for the MHC-I loading as well as once bound to H-2D b , predisposing them for TCR binding. Our results also indicate that peptide position 6, key for interaction of H-2D b /gp33 with the TCR P14, takes a suboptimal conformation before as well as after binding to the TCR. Analyses of H-2D b in complex with APLs, in which position 6 was subjected to an l - to d -amino acid modification, revealed small conformational changes and comparable pMHC thermal stability. However, the l - to d -modification reduced significantly the binding to P14 even in the presence of the p3P modification. Our combined data highlight the sensitivity of the TCR for the conformational dynamics of pMHC and provide further tools to dissect and modulate TCR binding and immunogenicity via APLs.

Protein aggregation into amyloid fibrils is the archetype of aberrant biomolecular self-assembly processes, with more than 50 diseases associated that are mostly uncurable. Understanding aggregation mechanisms is thus of fundamental importance and goes in parallel with the characterization of the structures of the transient oligomers formed in the process. Oligomers have been proven elusive to high-resolution structural techniques, while the large sizes and long-time scales typical of aggregation processes have limited, so far, the use of computational methods. To surmount these limitations, we introduce here multi-eGO, an atomistic, hybrid structure-based model, that leveraging on the knowledge of monomers conformational dynamics and of fibril structures, can efficiently capture the essential structural and kinetics aspects of protein aggregation. Multi-eGO molecular dynamics simulations can describe the aggregation kinetics of thousands of monomers. The concentration dependence of the simulated kinetics, as well as the structural features of the resulting fibrils, are in qualitative agreement with in vitro experiments on an amyloidogenic peptide of Transthyretin, a protein responsible for one of the most common cardiac amyloidosis. Multi-eGO simulations allow to observe in time and at atomic resolution the formation of primary nuclei in a sea of transient lower order oligomers, to follow their growth and the subsequent secondary nucleation events, till the maturation of multiple fibrils. Multi-eGO, combined with the many experimental techniques deployed to study protein aggregation, can provide the structural basis needed to advance the design of molecules targeting amyloidogenic diseases.