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The J-domain proteins (JDP) form the largest protein family among cellular chaperones. In cooperation with the Hsp70 chaperone system, these co-chaperones orchestrate a plethora of distinct functions, including those that help maintain cellular proteostasis and development. JDPs evolved largely through the fusion of a J-domain with other protein subdomains. The highly conserved J-domain facilitates the binding and activation of Hsp70s. How JDPs (re)wire Hsp70 chaperone circuits and promote functional diversity remains insufficiently explained. Here, we discuss recent advances in our understanding of the JDP family with a focus on the regulation built around J-domains to ensure correct pairing and assembly of JDP-Hsp70 machineries that operate on different clientele under various cellular growth conditions.

In order for a protein to be able to function correctly it needs to adopt its proper fold. Because of this, protein quality control (PQC) is vital at every level of cellular function. The central protein in PQC networks across all kingdoms of life are the Hsp70 molecular chaperones. In my thesis I present work into the adaptability of the chaperone network during different types of stress, and the functional variability between highly homologous yeast Hsp70 chaperones. During heat stress, Hsp70s collaborate with other chaperones to sequester misfolded proteins into inclusion bodies and later resolve them. Hydrogen peroxide stress however requires additional activity from the peroxiredoxin Tsa1. Tsa1 is needed to recruit chaperones to misfolded proteins during oxidative stress. Interestingly, this is also true for stress caused by ageing, and increasing the level of Tsa1 can prolong the lifespan of yeast. The Stress Seventy subfamily A (Ssa1-4) is an important Hsp70 family in yeast. Loss of Ssa1 and Ssa2 decreases cellular viability and lifespan even though Ssa3 and Ssa4 remain. Overproduction of Ssa4 can restore many but not all of the functions usually carried out by Ssa1/2, and restore a full lifespan. We show that preventing proteins from misfolding or sequestering them into inclusion bodies is enough to ensure a full lifespan, while disaggregation of inclusion bodies is not required for longevity assurance. We also describe a novel, Hsp70-dependent site for sequestration of misfolded proteins around the yeast nucleolus that forms after heat shock. The site is also the basis for the asymmetric segregation of damaged proteins in the nucleus during cell division, in a manner that is distinct from previously described asymmetry pathways in yeast.

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1′s function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1′s functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1′s NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.

Hsp70 chaperones interact with substrate proteins in a coordinated fashion that is regulated by nucleotides and enhanced by assisting cochaperones. There are numerous homologues and isoforms of Hsp70 that participate in a wide variety of cellular functions. This diversity can facilitate adaption or specialization based on particular biological activity and location within the cell. In this review, we highlight two specialized binding partner proteins, Tim44 and IRE1, that interact with Hsp70 at the membrane in order to serve their respective roles in protein translocation and unfolded protein response signalling. Recent mechanistic data suggest analogy in the way the two Hsp70 homologues (BiP and mtHsp70) can bind and release from IRE1 and Tim44 upon substrate engagement. These shared mechanistic features may underlie how Hsp70 interacts with specialized binding partners and may extend our understanding of the mechanistic repertoire that Hsp70 chaperones possess.

Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins.

Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins.

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. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the beta subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the beta subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discriminate different substrate proteins might allow its activation when client proteins require remodeling. A.A. thanks the Basque Government for a Predoctoral Fellowship. The excellent technical assistance of N. Orozco is gratefully acknowledged. We also thank Mathias P. Mayer for the plasmid encoding the DnaK SBD. This work was supported by grants BFU2016-75983 (AEI/FEDER, UE) and IT709-13 (Basque Government).

Kar2p, an essential Hsp70 chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae, facilitates the transport and folding of nascent polypeptides within the endoplasmic reticulum lumen. The chaperone activity of Kar2p is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, namely Sil1p and Lhs1p. Here, we demonstrate that the binding requirements for Lhs1p are complex, requiring both the nucleotide binding domain plus the linker domain of Kar2p. In contrast, the IIB domain of Kar2p is sufficient for binding of Sil1p, and point mutations within IIB specifically blocked Sil1p-dependent activation while remaining competent for activation by Lhs1p. Taken together, these results demonstrate that the interactions between Kar2p and its two nucleotide exchange factors can be functionally resolved and are thus mechanistically distinct.

13 páginas, 6 figuras.-- et al., The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of α-synuclein (αSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides. We show a nucleotide-dependent interaction between Hsp70 and αSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with αSyn. Such a co-aggregation phenomenon can be prevented in vitro by the co-chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of αSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration., CR and AA held FEBS Long-Term Fellowships. CWB is an EMBO Long-Term Postdoctoral Fellow, ATvdG was the recipient of a Topmaster fellowship of the graduate school GUIDE for Drug Exploration of the University of Groningen. STDH is a recipient of a Human Frontier Science Program Long-termFellowship (LT0798/ 2005) and is supported in part by the National Science Council of the Republic of China, Taiwan(NSC97-2917-1-564-102). JC is recipient of a Human Frontier Young Investigators Award (RGY67/ 2007) and also thanks the BBSRC (9015651/1). CMD and JC acknowledge funding from The Wellcome Trust and The Leverhulme Trust. DP is grateful to The Spanish Ministry of Health (PI05/2056; PI06/1641), The Spanish Ministry of Science and Innovation (SAF2007-29418E) and the PAIDI Program from the Regional Government (BIO323) for funding. EAAN acknowledges ZonMW Research Institute of Diseases in the Elderly and De Nederlandse Hersenstichting for funding.

You need the Java runtime environment to see the additional information in this article as Protein Structures., Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p–ScOrc 6p). Here, we report that a hydrophobic sequence (IL4) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p2–DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL4 motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL4 selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL4 results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL4 motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits., This work has been financed by Spanish MICINN (grants BFU2006-00494 and BIO2009-06952) and CAM (GR/SAL/0651/2004) to R.G. and MICINN (BFU2007-62382) and the EU (“3D repertoire” LSHG-CT-2005-512028) to J.M.V.

The bacterial chaperone SecB assists translocation of proteins across the inner membrane. The mechanism by which it differentiates between secretory and cytosolic proteins is poorly understood. To identify its binding motif, we screened 2688 peptides covering sequences of 23 proteins for SecB binding. The motif is approximately 9 residues long and is enriched in aromatic and basic residues, whereas acidic residues are disfavored. Its identification allows the prediction of binding regions within protein sequences with up to 87% accuracy. SecB-binding regions occur statistically every 20-30 residues. The occurrence and affinity of binding regions are similar in SecB-dependent and -independent secretory proteins and in cytosolic proteins, and SecB lacks specificity toward signal sequences. SecB cannot thus differentiate between secretory and non-secretory proteins via its binding specificity. This conclusion is supported by the finding that SecB binds denatured luciferase, thereby allowing subsequent refolding by the DnaK system. SecB may rather be a general chaperone whose involvement in translocation is mediated by interactions of SecB and signal sequences of SecB-bound preproteins with the translocation apparatus.

Boosted by the technological advances in experimental techniques, cellular biology is nowadays facing the need for quantitative approaches in order to rationalize the huge amount of collected data. A particularly succesfull theoretical framework is provided by Polymer Theory which, combined with molecular simulations, can capture the essential features of biomacromolecules and describe the cellular processes they participate to. This thesis provides a compendium of works showing the strength of this combination. In a first project, we model the twisting properties of amyloid fibrils by means of a simple coarse-grained approach, based on the competition between elasticity and electrostatic repulsion of nearby portions of the fibrils. The model quantitatively recapitulates the evolution of fibril periodicity as a function of the ionic strength of the solution and of the fibril width. A universal mesoscopic structural signature of the fibrils emerges from this picture, predicting a general, parameter-free law for the periodicity of the fibrils which is validated on several experimental results. A second work is focused on the role played by mitochondrial Hsp70 chaperone in the import of cytoplasmic proteins. Particularly, we computed by means of molecular simulations the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process and outline the essential role played by Hsp70 in this context. Finally, in a third project we studied the shape properties of a polymer under tension, a physical condition typically realized both in single-molecule experiments and in vivo. By means of analytical calculations and Monte Carlo simulations, we develop a theoretical framework which quantitatively describes these properties, highlighting the interplay between external force and chain size in determining the spatial distribution of a stretched chain.