Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins1-3. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity4-9. GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p2310. However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70-Hsp90 'client-loading complex' and an activated Hsp90-p23 'client-maturation complex' is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR-Hsp90-p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase-Hsp90 structure11. Thus, aided by direct co-chaperone-client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure12, we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge, complete chaperone cycle for any Hsp90 client.

Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function.

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an "ADP-like" undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70-Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.

Efficient targeting of Hsp70 chaperones to substrate proteins depends on J-domain cochaperones, which in synergism with substrates trigger ATP hydrolysis in Hsp70s and concomitant substrate trapping. We present the crystal structure of the J-domain of Escherichia coli DnaJ in complex with the E. coli Hsp70 DnaK. The J-domain interacts not only with DnaK's nucleotide-binding domain (NBD) but also with its substrate-binding domain (SBD) and packs against the highly conserved interdomain linker. Mutational replacement of contacts between J-domain and SBD strongly reduces the ability of substrates to stimulate ATP hydrolysis in the presence of DnaJ and compromises viability at heat shock temperatures. Our data demonstrate that the J-domain and the substrate do not deliver completely independent signals for ATP hydrolysis, but the J-domain, in addition to its direct influence on Hsp70s catalytic center, makes Hsp70 more responsive for the hydrolysis-inducing signal of the substrate, resulting in efficient substrate trapping.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and ß-subdomain. We identified a flexible region within the rigid ß-subdomain that gives way under load, thus opening up the α/ß interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication.

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms.

Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain.

Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis.

Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved.

Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop.

In eukarya, chaperones Hsp70 and Hsp90 act coordinately in the folding and maturation of a range of key proteins with the help of several co-chaperones, especially Hop. Although biochemical data define the Hop-mediated Hsp70-Hsp90 substrate transfer mechanism, the intrinsic flexibility of these proteins and the dynamic nature of their complexes have limited the structural studies of this mechanism. Here we generate several complexes in the Hsp70/Hsp90 folding pathway (Hsp90:Hop, Hsp90:Hop:Hsp70 and Hsp90:Hop:Hsp70 with a fragment of the client protein glucocorticoid receptor (GR-LBD)), and determine their 3D structure using electron microscopy techniques. Our results show that one Hop molecule binds to one side of the Hsp90 dimer in both extended and compact conformations, through Hop domain rearrangement that take place when Hsp70 or Hsp70:GR-LBD bind to Hsp90:Hop. The compact conformation of the Hsp90:Hop:Hsp70:GR-LBD complex shows that GR-LBD binds to the side of the Hsp90 dimer opposite the Hop attachment site.

ATPase subdomain IA is a mediator of interdomain allostery in Hsp70 molecular chaperones.

The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other's activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the ß-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions.

Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays.

Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. BIOLOGICAL SIGNIFICANCE: Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Single-cell imaging of the heat-shock response in colon cancer cells suggests that magnitude and length rather than time of onset determines resistance to apoptosis.

Targeting the proteasome is a valuable approach for cancer therapy, potentially limited by pro-survival pathways that are induced in parallel to cell death. Whether these pro-survival pathways are activated in all cells, show different activation kinetics in sensitive versus resistant cells or interact functionally with cell death pathways is unknown. We monitored activation of the heat-shock response (HSR), a key survival pathway induced by proteasome inhibition, relative to apoptosis activation in HCT116 colon cancer cells expressing enhanced green fluorescent protein (EGFP) under the control of the HSP70 promoter. Single-cell and high-content time-lapse imaging of epoxomicin treatment revealed that neither basal activity nor the time of onset of the HSR differed between resistant and sensitive populations. However, resistant cells had significantly higher and prolonged reporter activity than those that succumbed to cell death. p53 deficiency protected against cell death but failed to modulate the HSR. By contrast, inhibition of the HSR significantly increased the cytotoxicity of epoxomicin. Our data provide novel insights into the kinetics and heterogeneity of the HSR during proteasome inhibition, suggesting that the HSR modulates cell death signalling unidirectionally.

Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry.

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether, LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action.

Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome.

No-go decay (NGD) is a mRNA quality-control mechanism in eukaryotic cells that leads to degradation of mRNAs stalled during translational elongation. The key factors triggering NGD are Dom34 and Hbs1. We used cryo-EM to visualize NGD intermediates resulting from binding of the Dom34-Hbs1 complex to stalled ribosomes. At subnanometer resolution, all domains of Dom34 and Hbs1 were identified, allowing the docking of crystal structures and homology models. Moreover, the close structural similarity of Dom34 and Hbs1 to eukaryotic release factors (eRFs) enabled us to propose a model for the ribosome-bound eRF1-eRF3 complex. Collectively, our data provide structural insights into how stalled mRNA is recognized on the ribosome and how the eRF complex can simultaneously recognize stop codons and catalyze peptide release.

Hsp10, Hsp70, and Hsp90 immunohistochemical levels change in ulcerative colitis after therapy.

Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) characterized by damage of large bowel mucosa and frequent extra-intestinal autoimmune comorbidities. The role played in IBD pathogenesis by molecular chaperones known to interact with components of the immune system involved in inflammation is unclear. We previously demonstrated that mucosal Hsp60 decreases in UC patients treated with conventional therapies (mesalazine, probiotics), suggesting that this chaperonin could be a reliable biomarker useful for monitoring response to treatment, and that it might play a role in pathogenesis. In the present work we investigated three other heat shock protein/molecular chaperones: Hsp10, Hsp70, and Hsp90. We found that the levels of these proteins are increased in UC patients at the time of diagnosis and decrease after therapy, supporting the notion that these proteins deserve attention in the study of the mechanisms that promote the development and maintenance of IBD, and as biomarkers of this disease (e.g., to monitor response to treatment at the histological level).

Effects of the disruption of the HSP70-II gene on the growth, morphology, and virulence of Leishmania infantum promastigotes

The 70-kDa heat shock protein (HSP70) is highly conserved among both prokaryotes and eukaryotes and plays essential roles in diverse cellular functions not only under stress but also under normal conditions. In the protozoan Leishmania infantum, the causative agent of visceral leishmaniasis, HSP70 is encoded by two HSP70 genes. Here, we describe the phenotypic alterations of HSP70-II-deficient (Deltahsp70-II) promastigotes. The absence of HSP70-II caused a major alteration in growth as the promastigotes reached stationary phase. In addition, aberrant forms were frequently observed in Deltahsp70-II mutant cultures. An accumulation of cells in the G2/M phase in cultures of the Deltahsp70-II mutant was determined by flow cytometry. Furthermore, Deltahsp70-II promastigotes showed a limited capacity of multiplication within macrophages, even though attachment to and uptake by macrophages did not differ significantly from the wild-type. Moreover, Deltahsp70-II was highly attenuated in BALB/c mouse experimental infections. In mutants re-expressing HSP70-II, the growth rate was restored, the normal morphology was recovered, and interactions with macrophages increased. However, promastigotes re-expressing HSP70-II did not recover their virulence. Overall, these data highlight the essential role played by HSP70-II expression in Leishmania virulence, pointing to this gene as a promising target for therapeutic interventions (AU)

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Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans Hsp70.

Hsp70 chaperones are composed of two domains; the 40 kDa N-terminal nucleotide-binding domain (NDB) and the 30 kDa C-terminal substrate-binding domain (SBD). Structures of the SBD from Escherichia coli homologues DnaK and HscA show it can be further divided into an 18 kDa beta-sandwich subdomain, which forms the hydrophobic binding pocket, and a 10 kDa C-terminal three-helix bundle that forms a lid over the binding pocket. Across prokaryotes and eukaryotes, the NBD and beta-sandwich subdomain are well conserved in both sequence and structure. The C-terminal subdomain is, however, more evolutionary variable and the only eukaryotic structure from rat Hsc70 revealed a diverged helix-loop-helix fold. We have solved the crystal structure of the C-terminal 10 kDa subdomain from Caenorhabditis elegans Hsp70 which forms a helical-bundle similar to the prokaryotic homologues. This provides the first confirmation of the structural conservation of this subdomain in eukaryotes. Comparison with the rat structure reveals a domain-swap dimerisation mechanism; however, the C. elegans subdomain exists exclusively as a monomer in solution in agreement with the hypothesis that regions out with the C-terminal subdomain are necessary for Hsp70 self-association.

Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro.

Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid beta (Abeta) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Abeta aggregation. These observations led to the hypothesis that chaperones might interact with Abeta and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Abeta has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Abeta aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Abeta self-assembly and that these chaperones are effective at substoichiometric concentrations (approximately 1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Abeta is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.

The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules.

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.

Heat stress enhances recovery of hepatocyte bile acid and organic anion transporters in endotoxemic rats by multiple mechanisms.

Heat stress (HS) reduces the many sequelae of lipopolysaccharide (LPS)-induced endotoxemia. Without HS, endotoxins have been shown to induce a transcriptional down-regulation of hepatocyte transport proteins for bile acids and organic anions. We performed experiments in isolated perfused rat livers at various times after LPS administration with and without HS pretreatment to determine whether HS would correct deficient transport of bromosulfophthalein (BSP). Possible mechanisms involved were investigated in livers from intact animals. In isolated perfused livers, LPS injection reduced BSP excretion to 48% compared with saline-injected controls (P < 0.01). When HS was applied 2 hours prior to LPS, BSP excretion increased to 74% of controls (P < 0.05 vs LPS and controls). Expression of the basolateral (Oatp1a1) and canalicular (Mrp2) organic anion transporter involved in the transport of BSP recovered more rapidly when HS preceded LPS application. Recovery of mRNA levels of these transporters occurred also earlier. Coimmunoprecipitation experiments and immunoelectron microscopy using a double immunogold labeling of heat shock protein 70 (HSP70) and various hepatocyte transporters suggested colocalization with HSP70 for the canalicular bile acid transporter (Bsep) in the subcanalicular space. In contrast, no colocalization was shown for Ntcp and anion transporters. In conclusion, we could show that HS enhances recovery of organic anion transporters and bile acid transporters following endotoxemia. Faster recovery of mRNA seems to be a key mechanism for anion transporters, whereas physical interaction with HSP70 plays a role in preservation of bile acid transporters. This interaction of HSP70 and canalicular transporters occurs only in pericanalicular vesicles but not when the protein is integrated into the plasma membrane.

Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering.

To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.