A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs.

Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis.

Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis.

Role of J-domain Proteins in Yeast Physiology and Protein Quality Control.

The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the folding of newly synthesized proteins to the disassembly and refolding of protein aggregates. This multifunctionality of Hsp70 is governed by J-domain proteins (JDPs), which act as indispensable co-chaperones that target specific substrates to Hsp70. The number of distinct JDPs present in a species always outnumbers Hsp70, documenting JDP function in functional diversification of Hsp70.

J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.

The processing, membrane targeting and folding of newly synthesized polypeptides is closely linked to their synthesis at the ribosome. A network of enzymes, chaperones and targeting factors engages ribosome-nascent chain complexes (RNCs) to support these maturation processes. Exploring the modes of action of this machinery is critical for our understanding of functional protein biogenesis.

Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources.

The cytoprotective sequestration activity of small heat shock proteins is evolutionarily conserved.

The chaperone-mediated sequestration of misfolded proteins into inclusions is a pivotal cellular strategy to maintain proteostasis in Saccharomyces cerevisiae, executed by small heat shock proteins (sHsps) Hsp42 and Btn2. Direct homologs of Hsp42 and Btn2 are absent in other organisms, questioning whether sequestration represents a conserved proteostasis strategy and, if so, which factors are involved. We examined sHsps from Escherchia coli, Caenorhabditis elegans, and humans for their ability to complement the defects of yeast sequestrase mutants.

Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase.

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly.

Bacterial ribosome collision sensing by a MutS DNA repair ATPase paralogue.

Ribosome stalling during translation is detrimental to cellular fitness, but how this is sensed and elicits recycling of ribosomal subunits and quality control of associated mRNA and incomplete nascent chains is poorly understood. Here we uncover Bacillus subtilis MutS2, a member of the conserved MutS family of ATPases that function in DNA mismatch repair, as an unexpected ribosome-binding protein with an essential function in translational quality control. Cryo-electron microscopy analysis of affinity-purified native complexes shows that MutS2 functions in sensing collisions between stalled and translating ribosomes and suggests how ribosome collisions can serve as platforms to deploy downstream processes: MutS2 has an RNA endonuclease small MutS-related (SMR) domain, as well as an ATPase/clamp domain that is properly positioned to promote ribosomal subunit dissociation, which is a requirement both for ribosome recycling and for initiation of ribosome-associated protein quality control (RQC).

Combinations of slow-translating codon clusters can increase mRNA half-life in .

The presence of a single cluster of nonoptimal codons was found to decrease a transcript's half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in The impact of multiple nonoptimal codon clusters on a transcript's half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5' end can lead to synergistic effects that increase a messenger RNA's (mRNA's) half-life in Specifically, the 5' end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold.

The Diverse Functions of Small Heat Shock Proteins in the Proteostasis Network.

The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and refolding activities executed by ATP-dependent proteases and chaperones constitute major strategies of the proteostasis network. Small heat shock proteins represent ATP-independent chaperones that bind to misfolded proteins, preventing their uncontrolled aggregation.

N-terminal acetylation of proteins by NatA and NatB serves distinct physiological roles in Saccharomyces cerevisiae.

N-terminal (Nt) acetylation is a highly prevalent co-translational protein modification in eukaryotes, catalyzed by at least five Nt acetyltransferases (Nats) with differing specificities. Nt acetylation has been implicated in protein quality control, but its broad biological significance remains elusive. We investigate the roles of the two major Nats of S.

Interactions between nascent proteins translated by adjacent ribosomes drive homomer assembly.

Accurate assembly of newly synthesized proteins into functional oligomers is crucial for cell activity. In this study, we investigated whether direct interaction of two nascent proteins, emerging from nearby ribosomes (co-co assembly), constitutes a general mechanism for oligomer formation. We used proteome-wide screening to detect nascent chain-connected ribosome pairs and identified hundreds of homomer subunits that co-co assemble in human cells.

HSP40 proteins use class-specific regulation to drive HSP70 functional diversity.

The ubiquitous heat shock protein 70 (HSP70) family consists of ATP-dependent molecular chaperones, which perform numerous cellular functions that affect almost all aspects of the protein life cycle from synthesis to degradation. Achieving this broad spectrum of functions requires precise regulation of HSP70 activity. Proteins of the HSP40 family, also known as J-domain proteins (JDPs), have a key role in this process by preselecting substrates for transfer to their HSP70 partners and by stimulating the ATP hydrolysis of HSP70, leading to stable substrate binding.

Molecular dissection of amyloid disaggregation by human HSP70.

The deposition of highly ordered fibrillar-type aggregates into inclusion bodies is a hallmark of neurodegenerative diseases such as Parkinson's disease. The high stability of such amyloid fibril aggregates makes them challenging substrates for the cellular protein quality-control machinery. However, the human HSP70 chaperone and its co-chaperones DNAJB1 and HSP110 can dissolve preformed fibrils of the Parkinson's disease-linked presynaptic protein α-synuclein in vitro.

Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates.

Variation in translation-elongation kinetics along a transcript's coding sequence plays an important role in the maintenance of cellular protein homeostasis by regulating co-translational protein folding, localization, and maturation. Translation-elongation speed is influenced by molecular factors within mRNA and protein sequences. For example, the presence of proline in the ribosome's P- or A-site slows down translation, but the effect of other pairs of amino acids, in the context of all 400 possible pairs, has not been characterized.

DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4.

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR.

Selective 40S Footprinting Reveals Cap-Tethered Ribosome Scanning in Human Cells.

Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave.

Chaperone-Mediated Protein Disaggregation Triggers Proteolytic Clearance of Intra-nuclear Protein Inclusions.

The formation of insoluble inclusions in the cytosol and nucleus is associated with impaired protein homeostasis and is a hallmark of several neurodegenerative diseases. Due to the absence of the autophagic machinery, nuclear protein aggregates require a solubilization step preceding degradation by the 26S proteasome. Using yeast, we identify a nuclear protein quality control pathway required for the clearance of protein aggregates.

Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species.

The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here, we show that an ATP-dependent human chaperone system disassembles Tau fibrils We found that this function is mediated by the core chaperone HSC70, assisted by specific cochaperones, in particular class B J-domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF).

The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species.

The accumulation and prion-like propagation of α-synuclein and other amyloidogenic proteins are associated with devastating neurodegenerative diseases. Metazoan heat shock protein HSP70 and its co-chaperones DNAJB1 and HSP110 constitute a disaggregation machinery that is able to disassemble α-synuclein fibrils in vitro, but its physiological effects on α-synuclein toxicity are unknown. Here, we depleted Caenorhabditis elegans HSP-110 and monitored the consequences on α-synuclein-related pathological phenotypes such as misfolding, intercellular spreading, and toxicity in C.

Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones.

Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant.

Publisher Correction: Processive extrusion of polypeptide loops by a Hsp100 disaggregase.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.