Amyloid fibrils, crystal-like aggregates of denatured proteins, form upon breakdown of supersaturation and underlie amyloidosis in diseases, such as Alzheimer's, Parkinson's, and amyloidogenic light chain (AL) disease. In AL amyloidosis, fibrils accumulate in the heart, where they are exposed to shear stress. To mimic cardiac amyloidosis, where each heartbeat causes diameter fluctuations in coronary arterioles, we employ a peristaltic pump using the same mechanical principle to induce shear stress, combined with thioflavin T fluorescence microscopy for in situ real-time observation of amyloid formation of an AL protein associated with heart amyloidosis. Our results demonstrate that confined geometries amplify shear stress-dependent amyloid nucleation. Furthermore, using grid-type microchannels to model arteriole networks under shear stress, we observed fluorescent amyloid bursts reflecting dynamic pathways shaped by fibril deposition, local mechanical stress, and channel occlusion. We also observed that mechanical agitation using a micromanipulator, in conjunction with disaggregating agents, was effective for removing amyloid deposits. Collectively, our findings reveal key principles underlying shear stress-dependent amyloid formation and provide a novel framework for understanding amyloid deposition in organs, potentially advancing anti-amyloidosis strategies.

Binding of the surrogate light chain (SLC) to the heavy chain (HC) of the pre-B cell receptor (preBCR) is an important quality control checkpoint during B cell development as roughly 50% of the rearranged HCs are defective. Unlike the regular light chain (LC), the SLC is a hetero-dimer of VpreB and λ5, both containing unstructured extensions, the unique regions. The molecular mechanisms that underlie the complex assembly processes which give rise to the final pre-BCR is not fully understood. Here we show, via reconstitution of the pre-BCR in vitro and in cells that λ5 plays a key role in the pre-BCR assembly. During SLC assembly, a β-strand, located between the λ5 domain and the unique region, induces structure in the largely unfolded VpreB, creating a high affinity complex. In addition, association of λ5 with the unstructured HC CH1 domain is required for its folding. This is essential for pre-BCR assembly and its release from the endoplasmic reticulum (ER). Finally, the unique region of λ5 plays a pivotal role in the antigen interaction of the SLC-HC complex. Together, our results reveal a multi-step mechanism for SLC and pre-BCR assembly, governed by association-induced folding reactions required for structural integrity and function.

The molecular chaperone Hsp90 is a major protein folding factor in the cytosol of eukaryotic cells. Its conformational cycle is regulated by various co-chaperones and post-translational modifications (PTMs) such as phosphorylation. Most of the phosphorylation sites are conserved between the 2 isoforms of human Hsp90, Hsp90α and Hsp90β. The analysis of the function of these sites has revealed general functional principles of Hsp90. To what extent isoform-specific phosphorylation regulates Hsp90 function is less well understood. Here, we explore the effect of the phosphorylation of a residue (threonine 446) specific for the constitutionally active isoform Hsp90β. Since T446 is bioinformatically predicted to be phosphorylated by kinases regulating the intrinsic stress response (ISR), this modification links Hsp90 function to the ISR status of the cell in an isotype-specific manner which renders Hsp90 unresponsive to the co-chaperone-mediated modulation of its ATPase activity and consequently affects client maturation. Hsp90β reverts to baseline ATPase-driven chaperoning activity which is no longer intensively regulated by co-chaperones and results in the rewiring of Hsp90-mediated protein quality control.

The diphthamide modification of eukaryotic translation elongation factor (eEF2) is important for accurate protein synthesis. While the enzymes for diphthamide synthesis are known, coordination of eEF2 synthesis with the diphthamide modification to maintain only modified eEF2 is unknown. Physical and genetic interactions extracted from BioGRID show a connection between diphthamide synthesis enzymes and chaperones in yeast. This includes the Hsp90 co-chaperones Hgh1 and Cpr7. The respective co-chaperone deletion strains contained eEF2 without diphthamide. Notably, strains deficient in other co-chaperones showed no defect in the eEF2-diphthamide modification. Our results demonstrate that diphthamide synthesis involves not only Dph enzymes but also the eEF2-interacting co-chaperones Hgh1 and Cpr7 and may thus require a conformational state of eEF2 which is maintained by specific (co-)chaperones.

The Hsp90 molecular chaperone system is regulated by numerous co-chaperones that modulate its function. In Saccharomyces cerevisiae, most of these cofactors can be deleted without affecting viability. Of the three essential ones, only the function of Sgt1 has remained enigmatic. Our in vivo and in vitro experiments define key structural elements and determine the essential function of Sgt1 in the chaperoning of client proteins. We demonstrate that yeast Sgt1 adopts a unique binding mode, engaging primarily with the middle domain of Hsp90. Through simultaneous interaction with both Hsp90 and client proteins, Sgt1 enhances client maturation efficiency. Specifically, Sgt1 stabilizes Hsp90-client complexes and prevents their dissociation by the co-chaperone Aha1. Our findings reveal a previously unrecognized layer of Hsp90 regulation, highlighting Sgt1 as a critical modulator of chaperone cycle progression.

Immunoglobulin M (IgM) antibodies are an essential and conserved part of adaptive immunity. IgMs assemble into pentamers and hexamers that bind to antigens with high avidity. Pentamers incorporate a small protein called J-chain (JC) that is important for their transcytosis via the poly-immunoglobulin receptor (pIgR). IgM antibodies can efficiently activate complement and interact with different Fc receptors (FcμR, Fcα/μR, pIgR) that trigger distinct effector functions and biodistribution. Even if these features have made the clinical use of IgM attractive over the past decades, there are currently no approved therapeutic IgMs on the market. In this review, we summarize the recent advances in the knowledge of IgM biogenesis and structure and discuss the therapeutic opportunities of IgM over IgG arising from high avidity, target clustering, binding to distinct IgM receptors, complement activation, transcytosis, and protein engineering opportunities. In addition, we summarize possibilities and outstanding challenges in the production of therapeutic IgM, including available technologies for IgM purification. Finally, we review recent preclinical and clinical data showing that IgM outperforms IgG in various in vitro assays but still fails to pass through clinical trials successfully. Challenges remain for IgM development, such as the need for a better understanding of IgM biology to facilitate a smoother transition from the preclinic to successful clinical trials.

The molecular chaperone Hsp90 is the central element of a chaperone machinery in the cytosol of eukaryotic cells that is characterized by a large number of structurally and functionally different co-chaperones that influence the core chaperone component in different ways and increase its influence on the proteome. From yeast to humans, the number of Hsp90 co-chaperones has increased from 14 to over 40, and new co-chaperones are still being discovered. While Hsp90 itself has only undergone limited changes in structure and mechanism from yeast to humans, its increased importance and contribution to different processes in humans is based on the evolution and expansion of the cohort of co-chaperones. In this review, we provide an overview of Hsp90 co-chaperones, focusing on their roles in regulating Hsp90 function and their evolution from yeast to humans.

The 2nd International Symposium on the Chaperone Code took place on October 26-28, 2023 at the Hilton Alexandria Old Town, VA, USA. The event featured more than 100 attendees from ten countries and provided a dynamic platform for established researchers, emerging investigators, postdoctoral fellows, and students to share insights and ideas on diverse facets of molecular chaperones with a strong focus on their regulation by post-translational modifications. The format fostered discussions and collaboration among participants. From the different contributions, future trajectories of the chaperone code field emerged, including avenues for further exploration and innovation in understanding and manipulating chaperone function in different diseases.

Deposition of amyloid plaques in the brains of Alzheimer's disease (AD) patients is a hallmark of the disease. AD plaques consist primarily of the beta-amyloid (Aβ) peptide but can contain other factors such as lipids, proteoglycans, and chaperones. So far, it is unclear how the cellular environment modulates fibril polymorphism and how differences in fibril structure affect cell viability. The small heat-shock protein (sHSP) alpha-B-Crystallin (αBC) is abundant in brains of AD patients, and colocalizes with Aβ amyloid plaques. Using solid-state NMR spectroscopy, we show that the Aβ40 fibril seed structure is not replicated in the presence of the sHSP. αBC prevents the generation of a compact fibril structure and leads to the formation of a new polymorph with a dynamic N-terminus. We find that the N-terminal fuzzy coat and the stability of the C-terminal residues in the Aβ40 fibril core affect the chemical and thermodynamic stability of the fibrils and influence their seeding capacity. We believe that our results yield a better understanding of how sHSP, such as αBC, that are part of the cellular environment, can affect fibril structures related to cell degeneration in amyloid diseases.

Abstract Polymeric IgM immunoglobulins have high avidity for antigen and complement, and dominate primary antibody responses. They are produced either as assemblies of six µ2L2 subunits (i.e., hexamers), or as pentamers of two µ2L2 subunits and an additional protein termed J-chain (JC), which allows transcytosis across epithelia. The molecular mechanism of IgM assembly with the desired stoichiometry remained unknown. Here, we show in vitro and in cellula that JC outcompetes the sixth IgM subunit during assembly. Before insertion into IgM, JC exists as an ensemble of largely unstructured, protease-sensitive species with heterogeneous, non-native disulfide bonds. The J-chain interacts with the hydrophobic β-sheets selectively exposed by nascent pentamers. Completion of an amyloid-like core triggers JC folding and drives disulfide rearrangements that covalently stabilize JC-containing pentamers. In cells, the quality control factor ERp44 surveys IgM assembly and prevents the secretion of aberrant conformers. This mechanism allows the efficient production of high-avidity IgM for systemic or mucosal immunity.

The antigen-binding sites in conventional antibodies are formed by hypervariable complementarity-determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra-long CDR. The HCs bearing ultra-long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra-long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra-long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.

Heat-shock proteins (Hsps) control protein folding in cells to ensure newly sensitized polypeptide chains attain their native structures. Hsps have been adapted to perform a number of different functions via their interactions with different co-chaperone proteins. The aim of this study is to resolve the conformational dynamics of the human Hsp90 (hHsp90) system using single-molecule Förster Resonance Energy Transfer (smFRET). Hsp90 is a dimeric protein that shows opening and closing motions during its ATPase cycle.

The response to proteotoxic stresses such as heat shock allows organisms to maintain protein homeostasis under changing environmental conditions. We asked what happens if an organism can no longer react to cytosolic proteotoxic stress. To test this, we deleted or depleted, either individually or in combination, the stress-responsive transcription factors Msn2, Msn4, and Hsf1 in Saccharomyces cerevisiae. Our study reveals a combination of survival strategies, which together protect essential proteins. Msn2 and 4 broadly reprogram transcription, triggering the response to oxidative stress, as well as biosynthesis of the protective sugar trehalose and glycolytic enzymes, while Hsf1 mainly induces the synthesis of molecular chaperones and reverses the transcriptional response upon prolonged mild heat stress (adaptation).

Posttranslational modifications of Hsp90 are known to regulate its in-vivo chaperone functions. Here we demonstrate that the lysine acetylation-deacetylation dynamics of Hsp82 is a major determinant in DNA repair mediated by Rad51. We uncover that the deacetylated lysine 27 in Hsp82 dictates the formation of the Hsp82-Aha1-Rad51 complex, which is crucial for client maturation. Intriguingly, Aha1-Rad51 complex formation is not dependent on Hsp82 or its acetylation status; implying that Aha1-Rad51 association precedes the interaction with Hsp82. The DNA damage sensitivity of Hsp82 (K27Q/ K27R) mutants are epistatic to the loss of the (de)acetylase hda1Δ; reinforcing the importance of the reversible acetylation of Hsp82 at the K27 position. These findings underscore the significance of the crosstalk between a specific Hsp82 chaperone modification code and the cognate co-chaperones in a client-specific manner. Given the pivotal role that Rad51 plays during DNA repair in eukaryotes and particularly in cancer cells, targeting the Hda1-Hsp90 axis could be explored as a new therapeutic approach against cancer.

Hsp90 is a molecular chaperone of central importance for protein homeostasis in the cytosol of eukaryotic cells, with key functional and structural traits conserved from yeast to man. During evolution, Hsp90 has gained additional functional importance, leading to an increased number of interacting co-chaperones and client proteins. Here, we show that the overall conformational transitions coupled to the ATPase cycle of Hsp90 are conserved from yeast to humans, but cycle timing as well as the dynamics are significantly altered. In contrast to yeast Hsp90, the human Hsp90 is characterized by broad ensembles of conformational states, irrespective of the absence or presence of ATP. The differences in the ATPase rate and conformational transitions between yeast and human Hsp90 are based on two residues in otherwise conserved structural elements that are involved in triggering structural changes in response to ATP binding. The exchange of these two mutations allows swapping of the ATPase rate and of the conformational transitions between human and yeast Hsp90. Our combined results show that Hsp90 evolved to a protein with increased conformational dynamics that populates ensembles of different states with strong preferences for the N-terminally open, client-accepting states.