Chaperone Binding Research Articles

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding.

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-κB (NF-kB) and inhibitor κB (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Abstract 16683: Subcellular Changes in NF-κB p50-Dependent iNOS Regulation in Doxorubicin-Induced Cardiomyopathy (DIC)

A repeat use of doxorubicin (Dox), an anti-cancer drug, can cause heart failure in cancer patients. Dox-induced cardiac mitochondrial as well as ER damage due to oxidative and nitrosative stress (O/NS) has been widely studied. However, mechanism by which Dox exerts iNOS changes via mitochondrial damage and/or ER stress is yet to be elucidated. We investigated whether iNOS induced inflammatory changes in Dox exposed hearts involve mitochondrial and/or endoplasmic reticulum (ER) damage. A cumulative dose of Dox (15mg/kg B.W., ip) was given to Male Wistar rats. We noted an increase in cytC release in the cytoplasm due to O/NS indicating mitochondrial injury. Dox induced linking of chaperone binding immunoglobulin protein (Bip) to activating transcription factor 6 (ATF6) suggested an increased unfolded protein response (UPR) contributing to ER stress which was independent of upstream transcription factors inositol requiring enzyme 1 (IRE1) and protein kinase RNA-like ER kinase (PERK). ATF6-induced ER stress promoted NFκBp105/p50-induced expression of iNOS and NO production in the myocardium. The latter was associated with increased expression of TLR2 and TRAF2 together with NFκB105/50 expression suggesting that ER-stress promoted the inflammatory response in the myocardium. Co-localization of iNOS or TLR2 together with calnexin/grp78 (ER markers) but not with Tom20 (mitochondria marker) suggested that only ER stress promoted inflammatory response. The study suggests that inhibition of Bip binding to ATF6 may modulate ER-stress-mediated inflammation in DIC and presents a novel avenue for a development of the therapeutics for treatment of DIC.

Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space

SummaryThe exchange of metabolites between the mitochondrial matrix and the cytosol depends on β-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and β-barrel protein biogenesis. Our work reveals how a single mitochondrial “transfer-chaperone” system is able to guide α-helical and β-barrel membrane proteins in a “nascent chain-like” conformation through a ribosome-free compartment.

A computational method to characterize the missense mutations in the catalytic domain of GAA protein causing Pompe disease.

Pompe disease is an autosomal recessive lysosomal storage disease caused by acid α-glucosidase (GAA) deficiency, resulting in intralysosomal accumulation of glycogen, including cardiac, skeletal, and smooth muscle cells. The GAA gene is located on chromosome 17 (17q25.3), the GAA protein consists of 952 amino acids; of which 378 amino acids (347-726) falls within the catalytic domain of the protein and comprises of active sites (518 and 521) and binding sites (404, 600, 616, and 674). In this study, we used several computational tools to classify the missense mutations in the catalytic domain of GAA for their pathogenicity and stability. Eight missense mutations (R437C, G478R, N573H, Y575S, G605D, V642D, L705P, and L712P) were predicted to be pathogenic and destabilizing to the protein structure. These mutations were further subjected to phenotyping analysis using SNPeffect 4.0 to predict the chaperone binding sites and structural stability of the protein. The mutations R437C and G478R were found to compromise the chaperone-binding activity with GAA. Molecular docking analysis revealed that the G478R mutation to be more significant and hinders binding to the DNJ (Miglustat) compared with the R437C. Further molecular dynamic analysis for the two mutations demonstrated that the G478R mutation was acquired higher deviation, fluctuation, and lower compactness with decreased intramolecular hydrogen bonds compared to the mutant R437C. These data are expected to serve as a platform for drug design against Pompe disease and will serve as an ultimate tool for variant classification and interpretations.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the proteome of porcine granulosa cells

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment. The exposure of living organisms to TCDD may result in numerous disorders, including reproductive pathologies. By employing two-dimensional fluorescence difference gel electrophoresis we aimed to identify proteins potentially involved in the mechanism of TCDD action and toxicity in porcine granulosa cells. The porcine granulosa cells were treated with TCDD (100 nM) for 3, 12 or 24 h, and afterwards, cytoplasmic proteins were isolated and labeled with cyanines. Next, samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF MS analysis. A total of 75 differentially expressed protein spots (p < 0.05 and fold change ≥2.0) were found in granulosa cells treated with TCDD. After 3, 12 and 24 h of TCDD treatment, we were able to identify 29, 34 and 12 spots, respectively. Functional analysis showed that cytoskeletal proteins formed the largest class of proteins significantly affected by TCDD in all time points. We also demonstrated that most of the identified proteins were associated with the “structural constituent of cytoskeleton” and “chaperone binding” Gene Ontology categories. Based on the analysis of the porcine granulosa cell proteome, we demonstrated that TCDD may affect the ovarian follicle fate by the rearrangement of the cytoskeleton and extracellular matrix as well as the modulation of proteins important for the cellular response to stress. The results of the current study present an extended insight into the TCDD mechanism of action in porcine granulosa cells, providing new directions for future functional studies.

Implications of step-chilling on meat color investigated using proteome analysis of the sarcoplasmic protein fraction of beef longissimus lumborum muscle

In order to improve beef color and color stability, step-chilling (SC) was applied on excised bovine longissimus lumborum muscle, with chilling starting at 0–4°C for 5 h, then holding the temperature at 12–18°C for 6 h, followed by 0–4°C again until 24 h post-mortem. pH and temperature were measured during rigor on SC loins as well as those subjected to routine chilling (RC, 0–4°C, till 24 h post-mortem). Color L*, a*, b* values, metmyoglobin (MetMb) content, MetMb reducing ability (MRA) and NADH content were determined on samples aged for 1, 7, and 14 d. Sarcoplasmic proteome analysis was only conducted on d 1 samples. The results showed muscles subjected to SC maintained a temperature at around 15°C for 5 to 10 h post-mortem, and exhibited a slow temperature decline, but rapid pH decline. Beef steaks treated with SC had higher L*, a*, b* and chroma values than those of RC samples at 1 and 7 d chilled storage (0–4°C), while showing no significant difference for a*, b* and chroma values at d 14. The SC samples also exhibited a lower relative content of surface MetMb, higher MRA and NADH content, compared with RC beef steaks during storage, indicating the SC-treated beef showed an improved color stability. Eleven differential protein spots/nine proteins were identified by two-dimensional gel electrophoresis and mass spectrometry, and those proteins were mainly involved in redox, chaperone binding, metabolic and peroxidase activity. Oxidoreductases play a role in decreasing the oxidation-induced myoglobin oxidation and benefiting the production of NADH, and finally improving the colour of beef. Of these, pyruvate dehydrogenase E1 component subunit beta showed a positive correlation with color L*, a*, b* values and accounted for more than 60% of the variation in color values; this protein can be considered as a potential beef color biomarker. The present study provided valuable information for studies on the molecular mechanism of color improvement from step-chilling, as well as for identifying markers associated with beef color.

Cellular metabolism network of Bacillus thuringiensis related to erythromycin stress and degradation

Erythromycin is one of the most widely used macrolide antibiotics. To present a system-level understanding of erythromycin stress and degradation, proteome, phospholipids and membrane potentials were investigated after the erythromycin degradation. Bacillus thuringiensis could effectively remove 77% and degrade 53% of 1 µM erythromycin within 24 h. The 36 up-regulated and 22 down-regulated proteins were mainly involved in spore germination, chaperone and nucleic acid binding. Up-regulated ribose-phosphate pyrophosphokinase and ribosomal proteins confirmed that the synthesis of protein, DNA and RNA were enhanced after the erythromycin degradation. The reaction network of glycolysis/gluconeogenesis was activated, whereas, the activity of spore germination was decreased. The increased synthesis of phospholipids, especially, palmitoleic acid and oleic acid, altered the membrane permeability for erythromycin transport. Ribose-phosphate pyrophosphokinase and palmitoleic acid could be biomarkers to reflect erythromycin exposure. Lipids, disease, pyruvate metabolism and citrate cycle in human cells could be the target pathways influenced by erythromycin. The findings presented novel insights to the interaction among erythromycin stress, protein interaction and metabolism network, and provided a useful protocol for investigating cellular metabolism responses under pollutant stress.

Chaperone-assisted peptide exchange on MHC-I is driven by a negative allostery release cycle: Implications for a role of peptide-editing Molecular Chaperones in scrutinizing the peptide repertoire

Abstract Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with major histocompatibility complex class I (MHC-I) to promote the loading of antigenic peptides through a poorly understood mechanism. Here, we use solution Nuclear Magnetic Resonance (NMR) spectroscopy to probe TAPBPR-induced changes on MHC-I. Dynamic motions present in the empty MHC groove become progressively dampened with increasing peptide occupancy, while allosteric communication between the A- and F-pockets regulates a conformational switch located near the TAPBPR binding site, which is crucial for chaperone release from the complex. Our analysis of NMR data recorded for a range of TAPBPR complexes prepared with both murine H2 and human HLA alleles complements the recent X-ray structures to provide atomic-resolution mechanistic insights into the selection of optimal peptide sequences for the displayed antigen repertoire. In particular, our results show that negative allosteric coupling between the MHC groove and chaperone binding sites allows TAPBPR to proofread MHC molecules containing a range of different peptides. Since the affinity of incoming peptides for the empty groove is greatly reduced in the chaperone complex, (micromolar range, relative to nanomolar for the free MHC), these interactions can provide a mechanism for optimizing the peptide repertoire, where only the highest-affinity peptides can drive chaperone release. Finally, our results suggest that TAPBPR may promote the dissociation of tightly bound peptides from MHC molecules, thereby further scrutinizing the displayed repertoire. These findings imply a similar mechanism for the specificity and editing function of tapasin in the peptide-loading complex.

Intrinsic properties and plasma membrane trafficking route of Src family kinase SH4 domains sensitive to retargeting by HIV-1 Nef

The HIV type 1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network, whereas the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18-amino acid Src homology 4 (SH4) domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for trans-Golgi network retargeting by Nef as well as the importance of the amino acid at position 8 for determining Nef sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that, analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and that intrinsic properties define SH4 domains as cargo of this Nef-sensitive lipoprotein binding chaperone-GTPase transport cycle.

Reduction of the molecular chaperone binding immunoglobulin protein (BiP) accentuates the effect of aging on sleep-wake behavior.

Sleep and wake quality, quantity, and architecture become modified with aging. Sleep and wake quality decline coinciding with increased fragmentation of both states across aging. We have previously shown that this age-related decline in sleep-wake quality is associated with increased endoplasmic reticular (ER) stress and decreased expression of the major ER chaperone binding immunoglobulin protein (BiP). BiP, also known as glucose-regulated protein 78, plays a key role in controlling the cellular response to ER stress, acting as a regulator of a protein homeostatic signaling pathway known as the unfolded protein response. Induction of BiP during cellular stress is part of an adaptive prosurvival mechanism. Here, using mice heterozygous for BiP, we investigated the effect of reduced BiP expression on sleep-wake behavior across aging; complete knockdown of BiP is embryonic lethal. We report that BiP heterozygosity accentuates the aging sleep-wake phenotype. Sleep and wake fragmentation was more pronounced in the BiP heterozygotes across the 3 ages examined. In mice lacking 1 functional copy of BiP, we observed an age-related significant reduction in wake bout duration and increase in wake bout numbers during the active period, as well as an increase in non rapid eye movement and rapid eye movement bout numbers accompanied by reduced bout durations of both non rapid eye movement and rapid eye movement during the sleep period. In addition, we observed increased ER stress in orexin neurons and occurrence of aggregates immunopositive for orexin at the terminals and projections of orexin neurons in the middle-aged BiP heterozygotes. Taken together, our data indicate that a reduction in the molecular chaperone BiP impacts sleep architecture across aging and that orexin processing is likely to be affected.

Severe Burn-Induced Intestinal Epithelial Barrier Dysfunction Is Associated With Endoplasmic Reticulum Stress and Autophagy in Mice.

The disruption of intestinal barrier plays a vital role in the pathophysiological changes after severe burn injury, however, the underlying mechanisms are poorly understood. Severe burn causes the disruption of intestinal tight junction (TJ) barrier. Previous studies have shown that endoplasmic reticulum (ER) stress and autophagy are closely associated with the impairment of intestinal mucosa. Thus, we hypothesize that ER stress and autophagy are likely involved in burn injury-induced intestinal epithelial barrier dysfunction. Mice received a 30% total body surface area (TBSA) full-thickness burn, and were sacrificed at 0, 1, 2, 6, 12 and 24 h postburn. The results showed that intestinal permeability was increased significantly after burn injury, accompanied by the damage of mucosa and the alteration of TJ proteins. Severe burn induced ER stress, as indicated by increased intraluminal chaperone binding protein (BIP), CCAAT/enhancer-binding protein homologous protein (CHOP) and inositol-requiring enzyme 1(IRE1)/X-box binding protein 1 splicing (XBP1). Autophagy was activated after burn injury, as evidenced by the increase of autophagy related protein 5 (ATG5), Beclin 1 and LC3II/LC3I ratio and the decrease of p62. Besides, the number of autophagosomes was also increased after burn injury. The levels of p-PI3K(Ser191), p-PI3K(Ser262), p-AKT(Ser473), and p-mTOR were decreased postburn, suggesting that autophagy-related PI3K/AKT/mTOR pathway is involved in the intestinal epithelial barrier dysfunction following severe burn. In summary, severe burn injury induces the ER stress and autophagy in intestinal epithelia, leading to the disruption of intestinal barrier.

CacyBP/SIP, a Hsp90 binding chaperone, in cellular stress response.

CacyBP/SIP interacts with Hsp90 and is able to protect proteins from denaturation and/or aggregation induced by elevated temperature. In this work we studied the influence of different stress factors on CacyBP/SIP level in HEp-2 cells. We have found that H2O2 and radicicol treatment resulted in a significant increase (up to 40%) in the CacyBP/SIP level. We have also found that HEp-2 cells overexpressing CacyBP/SIP were more resistant to stress-induced death. Further studies have revealed that the Hsf1 transcription factor binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression under stress conditions. To check whether the CacyBP/SIP protein might play a role in stress responses in vivo, we analyzed its level in selected brain structures of control and stressed mice. We have found that the level of the CacyBP/SIP protein was higher in the thalamus/hypothalamus, hippocampus and brainstem of stressed mice. Thus, the presented results clearly indicate that CacyBP/SIP is involved in cellular stress response.

Cork-in-Bottle Occlusion of Fluoride Ion Channels by Crystallization Chaperones

SummaryCrystallization of dual-topology fluoride (Fluc) channels requires small, soluble crystallization chaperones known as monobodies, which act as primary crystal lattice contacts. Previous structures of Flucs have been solved in the presence of monobodies that inhibit fluoride currents in single-channel electrophysiological recordings. These structures have revealed two-fold symmetric, doubly bound arrangements, with one monobody on each side of the membrane. The combined electrophysiological and structural observations raise the possibility that chaperone binding allosterically closes the channel, altering the structure from its conducting form. To address this, we identify and solve the structure with a different monobody that only partially blocks fluoride currents. The structure of the channel-monobody complex is asymmetric, with monobody bound to one side of the channel only. The channel conformation is nearly identical on the bound and uncomplexed sides, and to all previously solved structures, providing direct structural evidence that monobody binding does not induce local structural changes.

The force-dependent mechanism of DnaK-mediated mechanical folding

It is well established that chaperones modulate the protein folding free-energy landscape. However, the molecular determinants underlying chaperone-mediated mechanical folding remain largely elusive, primarily because the force-extended unfolded conformation fundamentally differs from that characterized in biochemistry experiments. We use single-molecule force-clamp spectroscopy, combined with molecular dynamics simulations, to study the effect that the Hsp70 system has on the mechanical folding of three mechanically stiff model proteins. Our results demonstrate that, when working independently, DnaJ (Hsp40) and DnaK (Hsp70) work as holdases, blocking refolding by binding to distinct substrate conformations. Whereas DnaK binds to molten globule-like forms, DnaJ recognizes a cryptic sequence in the extended state in an unanticipated force-dependent manner. By contrast, the synergetic coupling of the Hsp70 system exhibits a marked foldase behavior. Our results offer unprecedented molecular and kinetic insights into the mechanisms by which mechanical force finely regulates chaperone binding, directly affecting protein elasticity.

Evolutionarily-Encoded Translation Kinetics Coordinate Co-Translational SSB Chaperone Binding in Yeast

Chaperones can bind to the ribosomes and nascent polypeptides co-translationally to assist protein folding. It was not known previously whether there is any interplay between chaperone binding and translation kinetics. To study this effect, we utilize the high-throughput transcriptome-wide quantitative data for translation kinetics and chaperone binding from ribosome profiling and selective ribosome profiling methods respectively. In vivo selective ribosome profiling has shown that yeast Hsp70 chaperone Ssb associates broadly with a major fraction of nascent proteins. Using the ribosome footprint densities as a measure of local translation rate, we find that mRNA segments within the ribosome are translated faster during periods of Ssb binding to the nascent polypeptides compared to when Ssb is not bound. The acceleration of translation is maintained even when Ssb is knocked out thus implying an inherent encoding of faster translation within the mRNA sequence. Testing for mRNA features that can slow translation, we find that mRNA segments translated by Ssb-engaged ribosomes are enriched for fast-translated codons (having higher cognate tRNA concentrations), are depleted for slowly translated codons, and contain fewer proline codons. In addition, mRNA segments located 1-15 nucleotides downstream of Ssb-bound ribosomes have reduced mRNA secondary structure. Finally, nascent chain segments located in the ribosome tunnel of Ssb-bound ribosomes have average numbers of positively charged residues but are enriched in negatively charged residues. Taken together, these evolutionarily-encoded mRNA and nascent chain features cause faster translation during Ssb binding. This finding is significant as any alteration of translation kinetics due to synonymous mutations can potentially disrupt efficient binding of Ssb leading to possible misfolding of the protein without any change in its sequence.

Enhanced Accumulation of BnA7HSP70 Molecular Chaperone Binding Protein Improves Tolerance to Drought Stress in Transgenic Brassica napus

Enhanced Accumulation of <i>BnA7HSP70</i> Molecular Chaperone Binding Protein Improves Tolerance to Drought Stress in Transgenic <i>Brassica napus</i>

Prefoldins in Archaea.

Molecular chaperones promote the correct folding of proteins in aggregation-prone cellular environments by stabilizing nascent polypeptide chains and providing appropriate folding conditions. Prefoldins (PFDs) are molecular chaperones found in archaea and eukaryotes, generally characterized by a unique jellyfish-like hexameric structure consisting of a rigid beta-barrel backbone with protrudingflexible coiled-coils. Unlike eukaryotic PFDs that mainly interact with cytoskeletal components, archaeal PFDs can stabilize a wide range of substrates; such versatility reflects PFD's role as a key element in archaeal chaperone systems, which often lack general nascent-chain binding chaperone components such as Hsp70. While archaeal PFDs mainly exist as hexameric complexes, their structural diversity ranges from tetramers to filamentous oligomers. PFDs bind and stabilize nonnative proteins using varying numbers of coiled-coils, and subsequently transfer the substrate to a group II chaperonin (CPN) for refolding. The distinct structure and specific function of archaeal PFDs have been exploited for a broad range of applications in biotechnology; furthermore, a filament-forming variant of PFD has been used to fabricate nanoscale architectures of defined shapes, demonstrating archaeal PFDs' potential applicability in nanotechnology.

Atomistic simulations and network-based modeling of the Hsp90-Cdc37 chaperone binding with Cdk4 client protein: A mechanism of chaperoning kinase clients by exploiting weak spots of intrinsically dynamic kinase domains.

A fundamental role of the Hsp90 and Cdc37 chaperones in mediating conformational development and activation of diverse protein kinase clients is essential in signal transduction. There has been increasing evidence that the Hsp90-Cdc37 system executes its chaperoning duties by recognizing conformational instability of kinase clients and modulating their folding landscapes. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have combined atomistic simulations with protein stability and network-based rigidity decomposition analyses to characterize dynamic factors underlying allosteric mechanism of the chaperone-kinase cycle and identify regulatory hotspots that control client recognition. Through comprehensive characterization of conformational dynamics and systematic identification of stabilization centers in the unbound and client- bound Hsp90 forms, we have simulated key stages of the allosteric mechanism, in which Hsp90 binding can induce instability and partial unfolding of Cdk4 client. Conformational landscapes of the Hsp90 and Cdk4 structures suggested that client binding can trigger coordinated dynamic changes and induce global rigidification of the Hsp90 inter-domain regions that is coupled with a concomitant increase in conformational flexibility of the kinase client. This process is allosteric in nature and can involve reciprocal dynamic exchanges that exert global effect on stability of the Hsp90 dimer, while promoting client instability. The network-based rigidity analysis and emulation of thermal unfolding of the Cdk4-cyclin D complex and Hsp90-Cdc37-Cdk4 complex revealed weak spots of kinase instability that are present in the native Cdk4 structure and are targeted by the chaperone during client recruitment. Our findings suggested that this mechanism may be exploited by the Hsp90-Cdc37 chaperone to recruit and protect intrinsically dynamic kinase clients from degradation. The results of this investigation are discussed and interpreted in the context of diverse experimental data, offering new insights into mechanisms of chaperone regulation and binding.

Calreticulin: Challenges Posed by the Intrinsically Disordered Nature of Calreticulin to the Study of Its Function.

Calreticulin is a Ca2+-binding chaperone protein, which resides mainly in the endoplasmic reticulum but also found in other cellular compartments including the plasma membrane. In addition to Ca2+, calreticulin binds and regulates almost all proteins and most of the mRNAs deciding their intracellular fate. The potential functions of calreticulin are so numerous that identification of all of them is becoming a nightmare. Still the recent discovery that patients affected by the Philadelphia-negative myeloproliferative disorders essential thrombocytemia or primary myelofibrosis not harboring JAK2 mutations carry instead calreticulin mutations disrupting its C-terminal domain has highlighted the clinical need to gain a deeper understanding of the biological activity of this protein. However, by contrast with other proteins, such as enzymes or transcription factors, the biological functions of which are strictly defined by a stable spatial structure imprinted by their amino acid sequence, calreticulin contains intrinsically disordered regions, the structure of which represents a highly dynamic conformational ensemble characterized by constant changes between several metastable conformations in response to a variety of environmental cues. This article will illustrate the Theory of calreticulin as an intrinsically disordered protein and discuss the Hypothesis that the dynamic conformational changes to which calreticulin may be subjected by environmental cues, by promoting or restricting the exposure of its active sites, may affect its function under normal and pathological conditions.

Polymer translocation through a nanopore in the presence of chaperones: A three dimensional MD simulation study

The chaperone assisted polymer translocation through a nanopore is studied using Langevin dynamics simulations in three dimensions. We investigate the effects of the polymer persistence length, the binding energy between the polymer and the chaperones and the chaperone concentration on the translocation. The results show that the probability of translocation increases and the mean translocation time decreases by increasing the chaperone concentration and the binding energy for all persistence lengths. The translocation probability also increases as the polymer persistence length increases, but the translocation process of stiffer polymers takes longer time compared with the flexible ones. Also, it is found that the strength of chaperone binding differently affects the translocation probabilities of flexible and stiff polymers. Dynamics of the translocation is described using the waiting time distribution of monomers for different persistence lengths. The obtained results can be exploited to understand some biological chaperone assisted translocations in which different conditions such as a variation in the value of temperature, pH or salt concentration change the chain flexibility.