The self-association equilibrium of DNAJA2 regulates its interaction with unfolded substrate proteins and with Hsc70.

J-domain proteins tune the specificity of Hsp70s, engaging them in precise functions. Despite their essential role, the structure and function of many J-domain proteins remain largely unknown. We explore human DNAJA2, finding that it reversibly forms highly-ordered, tubular structures that can be dissociated by Hsc70, the constitutively expressed Hsp70 isoform.

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase.

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2.

MLb-LDLr: A Machine Learning Model for Predicting the Pathogenicity of Missense Variants.

Untreated familial hypercholesterolemia (FH) leads to atherosclerosis and early cardiovascular disease. Mutations in the low-density lipoprotein receptor () gene constitute the major cause of FH, and the high number of mutations already described in the makes necessary cascade screening or in vitro functional characterization to provide a definitive diagnosis. Implementation of high-predicting capacity software constitutes a valuable approach for assessing pathogenicity of variants to help in the early diagnosis and management of FH disease.

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species.

α-synuclein aggregation is present in Parkinson's disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied.

Extraction and Refolding Determinants of Chaperone-Driven Aggregated Protein Reactivation.

Reactivation of protein aggregates plays a fundamental role in numerous situations, including essential cellular processes, hematological and neurological disorders, and biotechnological applications. The molecular details of the chaperone systems involved are known to a great extent but how the overall reactivation process is achieved has remained unclear. Here, we quantified reactivation over time through a predictive mechanistic model and identified the key parameters that control the overall dynamics.

Structural and functional insights on the roles of molecular chaperones in the mistargeting and aggregation phenotypes associated with primary hyperoxaluria type I.

To carry out their biological function in cells, proteins must be folded and targeted to the appropriate subcellular location. These processes are controlled by a vast collection of interacting proteins collectively known as the protein homeostasis network, in which molecular chaperones play a prominent role. Protein homeostasis can be impaired by inherited mutations leading to genetic diseases.

Regulation of Human Hsc70 ATPase and Chaperone Activities by Apg2: Role of the Acidic Subdomain.

Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells.

Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate.

The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling.

Structural changes induced by acidic pH in human apolipoprotein B-100.

Acidification in the endosome causes lipoprotein release by promoting a conformational change in the LDLR allowing its recycling and degradation of LDL. Notwithstanding conformational changes occurring in the LDLR have expanded considerably, structural changes occurring in LDL particles have not been fully explored yet. The objectives of the present work were to study structural changes occurring in apoB100 by infrared spectroscopy (IR) and also LDL size and morphology by dynamic light scattering (DLS) and electron microscopy (EM) at both pH 7.

Crowding Modulates the Conformation, Affinity, and Activity of the Components of the Bacterial Disaggregase Machinery.

Chaperone-mediated protein aggregate reactivation is a complex reaction that depends on the sequential association of molecular chaperones on their interaction with protein aggregates and on substrate refolding. This process could be modulated by the highly crowded intracellular environment, which is known to affect protein conformational change, enzymatic activity, and protein-protein interactions. Here, we report that molecular crowding shapes the chaperone activity of bacterial disaggregase composed of the DnaK system (DnaK, DnaJ, and GrpE) and the molecular motor ClpB.

Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity.

Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem.

The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear.

ClpB dynamics is driven by its ATPase cycle and regulated by the DnaK system and substrate proteins.

The hexameric AAA+ (ATPase associated with various cellular activities) chaperone ClpB reactivates protein aggregates in collaboration with the DnaK system. An intriguing aspect of ClpB function is that the active hexamer is unstable and therefore questions how this chaperone uses multiple rounds of ATP hydrolysis to translocate substrates through its central channel. In the present paper, we report the use of biochemical and fluorescence tools to explore ClpB dynamics under different experimental conditions.

Human LDL structural diversity studied by IR spectroscopy.

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques.

Low-density lipoprotein density determination by electric conductivity.

The predominance of small dense low-density lipoprotein (LDL) particles is associated with an increased risk of coronary heart disease. A simple but precise method has been developed, based on electrical conductivity of an isopycnic gradient of KBr, to obtain density values of human LDL fraction. The results obtained can distinguish LDL density populations and their subfractions from different patients.

Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B.

ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood.

Nucleotide utilization requirements that render ClpB active as a chaperone.

ClpB is a member of the AAA+ superfamily that forms a ring-shaped homohexamer. Each protomer contains two nucleotide binding domains arranged in two rings that hydrolyze ATP. We extend here previous studies on ClpB nucleotide utilization requirements by using an experimental approach that maximizes random incorporation of different subunits into the protein hexamer.