Using steered molecular dynamics to predict and assess Hsp70 substrate-binding domain mutants that alter prion propagation.

Genetic screens using Saccharomyces cerevisiae have identified an array of cytosolic Hsp70 mutants that are impaired in the ability to propagate the yeast [PSI(+)] prion. The best characterized of these mutants is the Ssa1 L483W mutant (so-called SSA1-21), which is located in the substrate-binding domain of the protein. However, biochemical analysis of some of these Hsp70 mutants has so far failed to provide major insight into the specific functional changes in Hsp70 that cause prion impairment. In order to gain a better understanding of the mechanism of Hsp70 impairment of prions we have taken an in silico approach and focused on the Escherichia coli Hsp70 ortholog DnaK. Using steered molecular dynamics simulations (SMD) we demonstrate that DnaK variant L484W (analogous to SSA1-21) is predicted to bind substrate more avidly than wild-type DnaK due to an increase in numbers of hydrogen bonds and hydrophobic interactions between chaperone and peptide. Additionally the presence of the larger tryptophan side chain is predicted to cause a conformational change in the peptide-binding domain that physically impairs substrate dissociation. The DnaK L484W variant in combination with some SSA1-21 phenotypic second-site suppressor mutations exhibits chaperone-substrate interactions that are similar to wild-type protein and this provides a rationale for the phenotypic suppression that is observed. Our computational analysis fits well with previous yeast genetics studies regarding the functionality of the Ssa1-21 protein and provides further evidence suggesting that manipulation of the Hsp70 ATPase cycle to favor the ADP/substrate-bound form impairs prion propagation. Furthermore, we demonstrate how SMD can be used as a computational tool for predicting Hsp70 peptide-binding domain mutants that impair prion propagation

The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes

Deciphering Functional Significance of Substrate-Binding Domain of Ssa1 on Heat-Shock Response and Prion Propagation

The Hsp70 (70kDa heat shock protein) is highly conserved in all species and has been implicated in a variety of important cellular functions such as heat shock response, prion propagation, protein folding and refolding, translocation across membranes and assembly of macromolecular complexes. A variety of evidence has accumulated to show that Hsp70 machinery is a key modulator of the stress response, such as heat shock and oxidant stress. Moreover, many human neurodegenerative diseases such as Alzheimer’s, Parkinson’s and the prion disease Creutzfeldt-Jacob Disease (CJD) are also intimately linked to Hsp70. Structurally, Hsp70 is comprised of two domains: nucleotide-binding domain (NBD) and substrate-binding domain (SBD). In this work, a well-established yeast system and a combination of computational biology, genetics, structural biology, biochemistry and molecular biology were utilized to decipher the role of the SBD of Hsp70 in regulation of heat shock response and [PSI+] prion propagation. It was found that mutations (F475S and L483W) located in a region termed β6-β7 dramatically decreased the stability of the SBD and the size of side chain contributes to maintain the hydrophobic core of SBD. Introduction of smaller amino acid side chains at residue 475, such as alanine and serine, resulted in temperature sensitivity and [PSI+] impairment. When the side chain of residue 475 is larger than Cysteine, no matter if it is polar or nonpolar (tyrosine and phenylalanine), thermotolerance and [PSI+] can be maintained. However, there is a limit to side-chain size as too large, such as tryptophan, Ssa1 will lose intrinsic function and fail to support cell viability. By contrast, residue 483 prefer to smaller size amino acid to remain the hydrophobic core of SBD. Therefore, mutations on those two residues easily disturb the integrity of SBD and thus promote the degradation of SBD in vitro and in vivo. Inter-domain communication between the NBD and SBD is affected through disruption of the important hydrophobic core and disturbance of a critical interface between the two domains. Disruption of the SBD structure abolishes repression of ATP hydrolysis of the NBD, reduces protein refolding activity and alters interactions with co-chaperones, especially Hsp104 and Hsp26, but decreased the interactions with Sup35. Degradation of the SBD in vivo is dependent on the action of vacuolar carboxypeptidase (Pep4) rather than the proteasome and occurs in WT cells at high temperature. Finally, SBD degradation is negatively regulated by the acetylation of four reversible hyperacetylated lysine residues, K86, K185, K354 and K562 of Ssa1. And, the thermotolerance regulation is independent from [PSI+] prion and alteration of the genome-wide translation caused by the read-through. The reversible hyperacetylated residues did not influence the basal expression level of the Hsp70 machinery, but rapidly respond the heat-shock stress by deacetylating themselves to stabilize SBD of Hsp70

Deciphering Functional Significance of Substrate-Binding Domain of Ssa1 on Heat-Shock Response and Prion Propagation

The Hsp70 (70kDa heat shock protein) is highly conserved in all species and has been implicated in a variety of important cellular functions such as heat shock response, prion propagation, protein folding and refolding, translocation across membranes and assembly of macromolecular complexes. A variety of evidence has accumulated to show that Hsp70 machinery is a key modulator of the stress response, such as heat shock and oxidant stress. Moreover, many human neurodegenerative diseases such as Alzheimer’s, Parkinson’s and the prion disease Creutzfeldt-Jacob Disease (CJD) are also intimately linked to Hsp70. Structurally, Hsp70 is comprised of two domains: nucleotide-binding domain (NBD) and substrate-binding domain (SBD). In this work, a well-established yeast system and a combination of computational biology, genetics, structural biology, biochemistry and molecular biology were utilized to decipher the role of the SBD of Hsp70 in regulation of heat shock response and [PSI+] prion propagation. It was found that mutations (F475S and L483W) located in a region termed β6-β7 dramatically decreased the stability of the SBD and the size of side chain contributes to maintain the hydrophobic core of SBD. Introduction of smaller amino acid side chains at residue 475, such as alanine and serine, resulted in temperature sensitivity and [PSI+] impairment. When the side chain of residue 475 is larger than Cysteine, no matter if it is polar or nonpolar (tyrosine and phenylalanine), thermotolerance and [PSI+] can be maintained. However, there is a limit to side-chain size as too large, such as tryptophan, Ssa1 will lose intrinsic function and fail to support cell viability. By contrast, residue 483 prefer to smaller size amino acid to remain the hydrophobic core of SBD. Therefore, mutations on those two residues easily disturb the integrity of SBD and thus promote the degradation of SBD in vitro and in vivo. Inter-domain communication between the NBD and SBD is affected through disruption of the important hydrophobic core and disturbance of a critical interface between the two domains. Disruption of the SBD structure abolishes repression of ATP hydrolysis of the NBD, reduces protein refolding activity and alters interactions with co-chaperones, especially Hsp104 and Hsp26, but decreased the interactions with Sup35. Degradation of the SBD in vivo is dependent on the action of vacuolar carboxypeptidase (Pep4) rather than the proteasome and occurs in WT cells at high temperature. Finally, SBD degradation is negatively regulated by the acetylation of four reversible hyperacetylated lysine residues, K86, K185, K354 and K562 of Ssa1. And, the thermotolerance regulation is independent from [PSI+] prion and alteration of the genome-wide translation caused by the read-through. The reversible hyperacetylated residues did not influence the basal expression level of the Hsp70 machinery, but rapidly respond the heat-shock stress by deacetylating themselves to stabilize SBD of Hsp70

Functional significance of Hsp70 post-translational modification in prion propagation and cellular function

The term prion (proteinaceous infectious particles) was first coined by Stanley Prusiner while naming the causative agent responsible for a group of invariably fatal neurodegenerative diseases collectively termed transmissible spongiform encephalopathies (TSE). A breakthrough in prion research came with the studies which revealed that yeast species Saccharomyces cerevisiae contains proteins that have the ability to form prions. Sup35 is a S. cerevisiae protein involved in termination of translation. In a prion state referred to as [PSI+], a significant portion of the Sup35 protein in the cell coalesces into non-functional, self-propagating, amyloid-like polymers. Thus, yeast strains that are [PSI+] show increased levels of nonsense suppression. Once present, [PSI+] propagates by recruitment of the soluble form of Sup35 into the aggregate in a manner analogous to that of mammalian prions. A search for genetic factors affecting propagation and maintenance of [PSI+] has identified an essential role for molecular chaperones, namely Hsp70 and Hsp104. The Hsp70 chaperone family and its associated co-chaperones are highly conserved from yeast to mammals. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. In the model eukaryotic S. cerevisiae, to efficiently carry out such functions Hsp70 works in concert with a number of co-chaperones to regulate ATPase hydrolysis cycle of Hsp70, which in-turn dictates the peptide-binding status of Hsp70. While much data has accrued in relation to the ATPase and substrate binding cycles of Hsp70 there is a distinct lack of information regarding the regulation of this important chaperone at the post-translational level. Recent global proteomic studies have demonstrated that in vivo Hsp70 is phosphorylated. Using a simplified yeast system this study systematically assessed a variety of non-phosphorylateable and phosphomimetic Hsp70 mutants for phenotypic alterations in Hsp70 functions. It was found that alteration of Hsp70 phosphorylation status in vivo can impair prion propagation, alter both basal and acquired thermotolerance and in some cases render cells inviable. By looking at analogous mutants in closely related cytosolic Hsp70s this study identified functional similarities and differences between highly homologous Hsp70 species. This study shows a clear link between Hsp70 phosphorylation status and in vivo function. Given Hsp70s central role in a variety of important cellular metabolic pathways and the conservation of these phosphorylatable sites in higher eukaryotes, these findings have far reaching implications

Molecular Dynamics Investigations of Structural Conversions in Transformer Proteins

Multifunctional proteins that undergo major structural changes to perform different functions are known as “Transformer Proteins”, which is a recently identified class of proteins. One such protein that shows a remarkable structural plasticity and has two distinct functions is the transcription antiterminator, RfaH. Depending on the interactions between its N-terminal domain and its C-terminal domain, the RfaH CTD exists as either an all-α-helix bundle or all-β-barrel structure. Another example of a transformer protein is the Ebola virus protein VP40 (eVP40), which exists in different conformations and oligomeric states (dimer, hexamer, and octamer), depending on the required function.I performed Molecular Dynamics (MD) computations to investigate the structural conversion of RfaH-CTD from its all-a to all-b form. I used various structural and statistical mechanics tools to identify important residues involved in controlling the conformational changes. In the full-length RfaH, the interdomain interactions were found to present the major barrier in the structural conversion of RfaH-CTD from all-a to all-b form. I mapped the energy landscape for the conformational changes by calculating the potential of mean force using the Adaptive Biasing Force and Jarzynski Equality methods. Similarly, the interdomain salt-bridges in the eVP40 protomer were found to play a critical role in domain association and plasma membrane (PM) assembly. This molecular dynamic simulation study is supported by virus like particle budding assays investigated by using live cell imaging that highlighted the important role of these saltbridges. I also investigated the plasma membrane association of the eVP40 dimer in various PM compositions and found that the eVP40 dimer readily associates with the PM containing POPS and PIP2 lipids. Also, the CTD helices were observed to be important in stabilizing the dimer-membrane complex. Coarse-grained MD simulations of the eVP40 hexamer and PM system revealed that the hexamer enhances the PIP2 lipid clustering at the lower leaflet of the PM. These results provide insight on the critical steps in the Ebola virus life cycle

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Analysis of mutations in superoxide dismutase-1 and the protective effects of heat shock proteins against mutant-SOD1 toxicity.

Genetic studies have revealed over 100 mutations in the gene encoding superoxide dismutase type-1 (SOD1) that cause Familial Amyotrophic Lateral Sclerosis (FALS). For the purpose of this thesis an in vitro model has been developed by stably over-expressing wild type (wt), G93A or G93R mutant SOD1 in neuronally derived ND7 cell line. It was found that wt-SOD1 could provide protection against a range of cell stresses including serum removal (plus retinoic acid) IFN-gamma, staurosporine camptothecin ischemia/reoxygenation glutamate and hydrogen peroxide (H2O2). In contrast, both mutant forms of SOD1 enhanced cell death with the G93R mutation being the more severe of the two mutations tested. Hence, the disease-associated mutations convert wt-SOD1 from a protective anti-apoptotic protein to a pro-apoptotic form. Most of the above stresses are FALS relevant stresses, which primarily induce apoptotic cell death in ND7 cells, implicated in FALS pathology. The neuroprotective effect of various heat shock proteins (Hsps) in the above system was studied, utilising a Herpes Simplex Virus (HSV) - based gene delivery system. For the first time, it was demonstrated that in an in vitro model of mutant-SOD1-induced toxicity, the exogenous expression of Hsp27 and/or Hsp70 protects both G93A and G93R-SOD1-mutant expressing cells, under all the stresses tested, and the dual expression of Hsp27 and Hsp70 is more effective in protecting against mutant-SOD1 cytotoxicity than either Hsp individually as assessed by trypan blue and TUNEL analysis. In addition, G93A and G93R- SOD1 mutant expressing cells were markedly protected by caspase-8 and caspase-9 inhibition. However no additive protective effect of Hsp and caspase inhibitor was observed. To further investigate the protective effect of wt-SOD1 and damaging effect of the mutant, an HSV-based gene delivery system was utilised. Primary cultures of dorsal root ganglia (DRGs) from postnatal rats, wild-type mice, transgenic Hsp27 and transgenic Hsp70 mice were infected with SOD1 viruses for wt-SOD1 and G93R-mutant SOD1 and subjected to stresses of NGF withdrawal, IFN-gamma and staurosporine treatment. Finally, the DRG experiments were repeated with additional delivery of Hsps via HSV vectors to further investigate the protective effect of the Hsps. These experiments provided further evidence on the protective role of Hsp27 and/or Hsp70 against mutant-SOD1 induced toxicity