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Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that inhibit amyloid fibril formation; however, their mechanisms of action remain poorly understood. sHSPs comprise a conserved α-crystallin domain flanked by variable N- and C-terminal regions. To investigate the functional contributions of these three regions, we compared the chaperone activities of various constructs of human αB-crystallin (HSPB5) and heat-shock 27-kDa protein (Hsp27, HSPB1) during amyloid formation by α-synuclein and apolipoprotein C-II. Using an array of approaches, including thioflavin T fluorescence assays and sedimentation analysis, we found that the N-terminal region of Hsp27 and the terminal regions of αB-crystallin are important for delaying amyloid fibril nucleation and for disaggregating mature apolipoprotein C-II fibrils. We further show that the terminal regions are required for stable fibril binding by both sHSPs and for mediating lateral fibril-fibril association, which sequesters preformed fibrils into large aggregates and is believed to have a cytoprotective function. We conclude that although the isolated α-crystallin domain retains some chaperone activity against amyloid formation, the flanking domains contribute additional and important chaperone activities, both in delaying amyloid formation and in mediating interactions of sHSPs with amyloid aggregates. Both these chaperone activities have significant implications for the pathogenesis and progression of diseases associated with amyloid deposition, such as Parkinson's and Alzheimer's diseases., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Selig et al.)

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively "misfolded" state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.

Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell.

Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin's F-actin bundling activity: when unphosphorylated, dematin's two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.

Amyloid deposits are proteinaceous extra-cellular aggregates associated with a diverse range of disease states. These deposits are composed predominantly of amyloid fibrils, the unbranched, beta-sheet rich structures that result from the misfolding and subsequent aggregation of many proteins. In addition, amyloid deposits contain a number of non-fibrillar components that interact with amyloid fibrils and are incorporated into the deposits in their native folded state. The influence of a number of the non-fibrillar components in amyloid-related diseases is well established; however, the mechanisms underlying these effects are poorly understood. Here we describe the effect of two of the most important non-fibrillar components, serum amyloid P component and apolipoprotein E, upon the solution behavior of amyloid fibrils in an in vitro model system. Using analytical ultracentrifugation, electron microscopy, and rheological measurements, we demonstrate that these non-fibrillar components cause soluble fibrils to condense into localized fibrillar aggregates with a greatly enhanced local density of fibril entanglements. These results suggest a possible mechanism for the observed role of non-fibrillar components as mediators of amyloid deposition and deposit stability.

Mixed lineage kinase 2 (MLK2) is a protein kinase that signals in the stress-activated Jun N-terminal kinase signal transduction pathway. We used immunoprecipitation and mass spectrometric analysis to identify MLK2-binding proteins in cell lines with inducible expression of green fluorescent protein-tagged MLK2. Here we report the identification of clathrin as a binding partner for MLK2 in both cultured cells and mammalian brain. We demonstrate that clathrin binding requires a motif (LLDMD) located near the MLK2 C terminus, which is similar to "clathrin box" motifs important for binding of clathrin coat assembly and accessory proteins to the clathrin heavy chain. A C-terminal fragment of MLK2 containing this motif binds strongly to clathrin, and mutation of the LLDMD sequence to LAAAD completely abrogates clathrin binding. We isolated clathrin-coated vesicles from green fluorescent protein-MLK2-expressing cells and from mouse brain lysates and found that MLK2 is enriched along with clathrin in these vesicles. In addition, we demonstrated that endogenous MLK2 co-immunoprecipitates with clathrin heavy chain from the vesicle-enriched fraction of mouse brain lysate. Furthermore, overexpression of MLK2 in cultured cells inhibits accumulation of labeled transferrin in recycling endosomes during receptor-mediated endocytosis. These findings suggest a role for MLK2 and the stress-signaling pathway at sites of clathrin activity in vesicle formation or trafficking.