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Expanding the family of extracellular chaperones: Identification of human plasma proteins with chaperone activity.

ABSTRACT: Proteostasis, the balance of protein synthesis, folding and degradation, is essential to maintain cellular function and viability, and the many known intracellular chaperones are recognized as playing key roles in sustaining life. In contrast, the identity of constitutively secreted extracellular chaperones (ECs) and their physiological roles in extracellular proteostasis is less completely understood. We designed and implemented a novel strategy, based on the well-known propensity of chaperones to bind to regions of hydrophobicity exposed on misfolding proteins, to discover new ECs present in human blood. We used a destabilized protein that misfolds at 37°C as "bait" to bind to potential ECs in human serum and captured the complexes formed on magnetic beads. Proteins eluted from the beads were identified by mass spectrometry and a group of seven abundant serum proteins was selected for in vitro analysis of chaperone activity. Five of these proteins were shown to specifically inhibit protein aggregation. Vitronectin and plasminogen activator-3 inhibited both the in vitro aggregation of the Alzheimer's β peptide (Aβ^1-42 ) to form fibrillar amyloid, and the aggregation of citrate synthase (CS) to form unstructured (amorphous) aggregates. In contrast, prothrombin, C1r, and C1s inhibited the aggregation of Aβ^1-42 but did not inhibit CS aggregation. This study thus identified five novel and abundant putative ECs which may play important roles in the maintenance of extracellular proteostasis, and which apparently have differing abilities to inhibit the amorphous and amyloid-forming protein aggregation pathways.

SUBMITTER: Geraghty NJ

PROVIDER: S-EPMC8521303 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Expanding the family of extracellular chaperones: Identification of human plasma proteins with chaperone activity.

Geraghty Nicholas J NJ Satapathy Sandeep S Kelly Megan M Cheng Flora F Lee Albert A Wilson Mark R MR

Protein science : a publication of the Protein Society 20211004 11

Proteostasis, the balance of protein synthesis, folding and degradation, is essential to maintain cellular function and viability, and the many known intracellular chaperones are recognized as playing key roles in sustaining life. In contrast, the identity of constitutively secreted extracellular chaperones (ECs) and their physiological roles in extracellular proteostasis is less completely understood. We designed and implemented a novel strategy, based on the well-known propensity of chaperones ...[more]

PMID: 34553437

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Project description:Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

Project description:The 70-kDa heat shock protein (HSP70) family of molecular chaperones represents one of the most ubiquitous classes of chaperones and is highly conserved in all organisms. Members of the HSP70 family control all aspects of cellular proteostasis such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes. These chaperones augment organismal survival and longevity in the face of proteotoxic stress by enhancing cell viability and facilitating protein damage repair. Extracellular HSP70s have a number of cytoprotective and immunomodulatory functions, the latter either in the context of facilitating the cross-presentation of immunogenic peptides via major histocompatibility complex (MHC) antigens or in the context of acting as "chaperokines" or stimulators of innate immune responses. Studies have linked the expression of HSP70s to several types of carcinoma, with Hsp70 expression being associated with therapeutic resistance, metastasis, and poor clinical outcome. In malignantly transformed cells, HSP70s protect cells from the proteotoxic stress associated with abnormally rapid proliferation, suppress cellular senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy. All of the cellular activities of HSP70s depend on their adenosine-5'-triphosphate (ATP)-regulated ability to interact with exposed hydrophobic surfaces of proteins. ATP hydrolysis and adenosine diphosphate (ADP)/ATP exchange are key events for substrate binding and Hsp70 release during folding of nascent polypeptides. Several proteins that bind to distinct subdomains of Hsp70 and consequently modulate the activity of the chaperone have been identified as HSP70 co-chaperones. This review focuses on the regulation, function, and relevance of the molecular Hsp70 chaperone machinery to disease and its potential as a therapeutic target.

Dataset Information

Expanding the family of extracellular chaperones: Identification of human plasma proteins with chaperone activity.

Publications

Expanding the family of extracellular chaperones: Identification of human plasma proteins with chaperone activity.

Similar Datasets

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