1,721,038 research outputs found
Mechanistic Insights Into Heat Shock Protein 90 (Hsp90) Trans-splicing in Giardia lamblia
No full textHsp90 gene is conserved and encoded by a single ORF with none to many cis-spliced introns across the biological kingdom. Previous studies from our lab have shown that Hsp90 gene in Giardia lamblia has a split nature having two ORFs present 777 kB apart on chromosome 5. The two ORFs transcribe independently to generate individual pre-mRNAs which get stitched by a novel trans-splicing mechanism to generate the mature full length Hsp90 mRNA.
In this study, we have reconstituted Hsp90 trans-splicing (Ts) reaction in vitro using purified pre-mRNA substrates with a view to understand the sequence elements and protein factors necessary for the trans-splicing reaction. We cloned partial sequences of HspN and HspC ORFs retaining the sequence elements such as 5’ splice site, 3’ splice site, branch point adenine, polypyrimidine tract and the 26 nucleotide complementary sequence elements which we have previously implicated in the trans-splicing of Hsp90 pre-mRNAs. Purified pre-mRNA substrates, in vitro transcribed from respective clones were investigated for their ability to undergo trans-splicing in vitro by employing three approaches namely Reverse-transcriptase polymerase chain reaction (RT-PCR), body labelled pre-mRNA based method and Northern blot to detect unique trans-spliced junction after incubation of pre-mRNAs in the Ts buffer in the presence and absence of nuclear extract. Interestingly, all three approaches confirmed the ability of pre-mRNAs to undergo self-splicing in vitro in a Mg2+ ion dependent manner.
We have further validated the presence of self-spliced junction sequence using Nanostring technology, which is quantitative, free from any amplification bias and has higher sensitivity. Nanostring technology employs the use of sequence specific DNA reporter probes chemically linked to unique fluorescent barcodes and unique capture probes to specifically hybridize and detect the target. To examine the presence of Ts reaction products (trans-spliced mRNA and lariat by-product), we designed probes to specifically target nucleotides unique to the products and thus distinguish them from reaction substrates. Nanostring technology validated the presence of Ts junctional sequence as well as presence of lariat by-product after the in vitro trans-splicing reaction of the pre-mRNAs.
In addition, we have demonstrated that the 26 nucleotide complementary sequences, a unique feature in these pre-mRNAs, are necessary as positioning elements for in vitro self-splicing reaction by employing oligo inhibition assay as well as using mutant pre-mRNA substrates in in vitro trans-splicing reaction. Further, to understand the importance of the critical sequence elements for the in vitro Ts reaction, we performed site directed mutagenesis to delete 5’ splice site-GT, 3’ splice site-AG, branch point adenine and polypyrimidine tract alone and in different combinations and investigated the ability of the mutant pre-mRNAs to undergo self-splicing. Analysis of the Ts reaction products resolved in high resolution PAGE post incubation of the wild type and the mutant pre-mRNAs in different combinations in the Ts buffer (devoid of nuclear proteins) revealed several incorrectly sized products as compared to wild type pre-mRNAs. Sequencing of these products uncovered that in the absence of the critical nucleotides previously implicated in trans-splicing reaction, other cryptic nucleotides participate in trans-esterification reaction resulting in incorrectly spliced products. Therefore, our study highlights the importance of these critical nucleotide elements to ensure accurate Hsp90 self-splicing reaction.
We performed in silico secondary structure analysis of HspN and HspC introns. Our structure prediction results for HspC intron highlighted similarity with crystal structure of Oceanobacillus Group II intron. HspN intron structure prediction highlighted structural reduction as compared to Oceanobacillus group II intron owing to compact HspN intron. Our results suggest that Hsp90 trans-splicing of Giardia lamblia may be mechanistically similar to Group II splicing with the limited repertoire of spliceosomal protein components identified in Giardia only facilitating the fidelity of trans-splicing reaction.
Furthermore, we have also addressed the in vivo mechanistic aspects of Hsp90 gene trans-splicing. We propose that Hsp90 trans-splicing reaction in vivo may be dictated by physical proximity of the corresponding pre-mRNAs. We have shown that HspN and HspC ORFs physically interact with each other using high resolution Chromosome Conformation Capture (3C) technology which accounts for interactions between specific loci on a population scale.
In addition to Hsp90, two other genes Dhcβ and Dhcγ undergo trans-splicing based expression from different ORFs scattered across different chromosomes of G. lamblia. A distinctive feature in the pre-mRNAs arising from fragmented genes of Hsp90, Dhcβ and Dhcγ dispersed several hundred kbs apart on the same chromosome and some on different chromosomes is the presence of complementary sequences in the split introns of pre-mRNAs to seek and base pair with corresponding pre-mRNAs. We conjectured that there could possibly be a specialized sub-compartment in Giardia nucleus where all split genes could be co-localized by chromatin looping. Our 3C results showed that HspN also interacts physically with Dhcβ C-2 present 1700 kb apart on chromosome 5 and Dhcγ C-1 on chromosome 3. We also experimentally determined the presence of Scaffold/Matrix attachment regions (S/MARs) in between the split genes on chromosome 5 which may aid tethering of chromatin loop to Giardia nuclear matrix.
Overall, our study highlights the role of cis acting elements in the pre-mRNAs and nuclear organization to facilitate trans-splicing of heat shock protein 90 (Hsp90) in Giardia lamblia.
-Vinithra Iyer (Prof. Utpal Tatu’s lab
Regulation of Flagellar Motility in Giardia lamblia and Trichomonas vaginalis
No full textProtozoan pathogens are responsible for infections that are highly prevalent, especially in developing and under-developed countries. Flagellar motility is exhibited by a variety of organisms ranging from bacteria to certain cell types in mammals. In the context of pathogens, flagellar motility plays a significant role in the establishment of infection in the host. Unlike bacterial motility, where the mechanism and its contribution to pathogenesis is well studied, flagellar motility in protozoan parasites is an ill-explored field. Understanding the regulation of motility in these organisms would highly enhance our understanding of pathogenesis in these organisms and may possibly open new avenues of interventions.
All eukaryotic flagella are made of microtubules and driven by dynein motor proteins. However, every organism is unique in terms of its flagellar waveform, beat frequency and its general motility pattern. In the current study, using biophysical and biochemical approach we studied the motility characteristics of Giardia lamblia and Trichomonas vaginalis, both flagellated protozoan pathogens which establish infection in the small intestine and the urogenital tract respectively. We have addressed the question of “how flagellar motility may be differentially regulated in an organelle as structurally conserved as the flagella”. We used a biophysical approach to first characterize the patterns of motility in both the organisms by combining microscopy and imaging with a high-speed camera to understand the movements of the cells to bring out the differences in terms of speed and beat frequency of the flagella. Our results demonstrate that despite overall conservation in flagellar structure, the pattern of tubulin post-translational modifications within the flagella are diverse and may contribute to variations in their patterns of motility. In this study, we have examined the tubulin post-translational modification in the protozoan parasites Giardia lamblia and Trichomonas vaginalis using global, untargeted mass spectrometry. Using cellular fractions generated by density gradient centrifugation we enriched flagellar fractions from the cell homogenate and examined the presence of novel post translational modifications by western blotting using specific antibodies as well as by mass spectrometry. Our results highlight organism specific 'tubulin code'. For example, we find that tubulin monoglycylation is a modification localized to the flagella present in G.lamblia but not so evident in T.vaginalis. We also showed the presence of glutamylated tubulin in both G.lamblia and T.vaginalis. Using MS/MS, we were also able to identify the previously unknown sites of monoglycylation in beta tubulin in G.lamblia as well as mono and tri glutamylation in T.vaginalis. Using isolated flagella, we also characterized the flagellar proteome in G.lamblia and T.vaginalis and identified 468 proteins in G.lamblia and 380 proteins in T.vaginalis flagella. The flagellar proteomes reveal unique mechanisms supporting energy generation required for flagellar motility in these organisms.
Altogether, the flagellar proteomes as well as the sites of tubulin PTMs in these organisms, our study reveals potential mechanisms in regulating flagellar motilities in these neglected protozoan parasites
Biochemical Characterization Of Heat Shock Protein 90 From Plasmodium Falciparum
Get PDFMolecular chaperones are a group of proteins which maintain cellular homeostasis by assisting de novo protein folding and their refolding to native state after destabilization due to external stress. They are also known as heat shock proteins as they were first discovered as a response to heat stress. It is now well established that the function of this group of proteins is not only restricted to protein homeostasis but also extends to diverse cellular processes such signal transduction, development and differentiation.
Heat shock protein 90 (Hsp90) is one of the most abundant molecular chaperones that is highly conserved from prokaryotes to eukaryotes. Hsp90 is an essential chaperone and is required for the viability of all eukaryotes examined so far including yeast, Drosophila and Caenorhabditis elegans. Hsp90 has emerged as an important regulator of cellular activities by virtue of its ability to interact with a diverse set of client proteins many of which include transcription factors, protein kinases and signaling molecules. Through interaction with these proteins it is involved in regulating cellular processes including growth, cell cycle, endocrine functions, apoptosis, differentiation and development. Further in Drosophila and plants, Hsp90 is thought to function as a capacitor for morphological evolution and phenotypic variation. Recently, it has also been implicated in the emergence of drug resistance in Candida albicans. Furthermore, the importance of Hsp90 in disease states, particularly in cancer, is strongly evident, where chaperoning of mutated and oncogenic proteins is critical for continuous proliferation of cells. This has led to the development of Hsp90 inhibitors as an anti-cancer drug. Geldanamycin (GA), a benzoquinone ansamycin was the first molecule shown to inhibit Hsp90 activity by binding to its ATP binding domain. A derivative of GA, 17-allylamino-17-demethoxygeldanamycin (17AAG), has shown promise in clinical studies and has entered Phase III clinical trials.
Hsp90 has been shown to be important for growth and development of many protozoan parasites. Inhibition of Hsp90 function in Leishmania, Emiera, Toxoplasma, Trypanosoma as well as Plasmodium causes a block in their developmental cycle. Previous studies from our laboratory have shown that inhibition of Hsp90 function prevents growth of malaria parasite in human erythrocytes in vitro. P. falciparum Hsp90 (PfHsp90) has also been shown to regulate parasite growth during the febrile episodes that are characteristic of malaria. While most of the studies highlighting the importance of PfHsp90 have relied on its pharmacological inhibition, its biochemical characterization and quantitative measurement of its interaction with GA in isolated system has not been explored. It was also not understood whether the in vitro model of Hsp90 inhibition could translate into inhibition of the parasite growth in an animal model of malaria. Since Hsp90 is a split ATPase requiring proper co-ordination between the residues on its N-terminal and middle domains, it would be desirable to biochemically characterize full length PfHsp90 to gain insights into its potential as an anti-malarial target.
The present study was initiated with an objective of understanding the biochemical properties of Hsp90 from P. falciparum in terms of ATP binding, ATP hydrolysis and its GA binding ability. We have also examined the potential of PfHsp90 to serve as a chemotherapeutic target using its clinically well-established inhibitor, 17AAG, in a preclinical mice model. Apart from using in vitro and in vivo models of malaria, we have also explored the efficacy of 17AAG in the P. falciparum samples collected from malaria patients. Additionally, we have examined the relevance of chaperones, in particular PfHsp90 in the samples collected from malaria patients. Finally, we have attempted to understand the unexplored biology of another malaria parasite P. vivax by a high throughput proteomics approach.
Biochemical characterization of PfHsp90 and its comparison with host Hsp90
Hsp90 belongs to GHKL (gyrase, Hsp90, histidine kinase, MutL) protein family having a characteristic novel ATP-binding Bergerat fold. The ATP binding pocket of GHKL family differs from the conventional nucleotide binding fold in the formation of a cone shaped pocket made up of four anti-parallel β-sheets and three α helices as opposed to parallel βsheets surrounded by α-helices in the latter. The most distinctive feature of Bergerat fold is the presence of ATP lid. Further, even within the GHKL family members the composition and the conformation of this ATP-lid differs, leading to different solvent exposure of the bound ATP. All Hsp90s from different organisms, characterized so far, have been shown to posses ATP binding and hydrolysis activity but so far PfHsp90 ATPase activity has not been characterized. Using intrinsic tryptophan fluorescence measurements, we found PfHsp90 to bind ATP with about 30% higher affinity than human Hsp90 (hHsp90). We further,
32 determined the ATPase activity of PfHsp90 by monitoring the direct conversion of (γ-P)
32-2 ATP to Pi. PfHsp90 bound and hydrolyzed ATP with a Km of 611 µM and kcat of 9.9 x 10
-1m . Interestingly, PfHsp90 showed six times higher ATPase activity as compared to its human homologue and more intriguingly the ATPase activity exhibited by PfHsp90 was highest among all the Hsp90s studied so far. Previous studies from our laboratory have provided sufficient evidence for inhibitory action of GA on Plasmodium growth inside the infected erythrocytes. GA is known to exert its inhibitory effect by binding to the ATP binding domain of Hsp90 thus inhibiting its chaperone activity. Earlier reports have shown that despite a high similarity between the ATP/GA binding region in Hsp90 from different organisms, there is a difference in their ability to bind GA. For example, in spite of all the hallmarks of ATP-binding pocket of Hsp90 family C. elegans Hsp90 does not bind GA. We have employed fluorescence spectroscopy to examine whether PfHsp90 can bind to GA. In parallel, we have also determined the binding affinity of human Hsp90 (hHsp90) to GA. We observed small but reproducible differences in the binding affinity of GA to Hsp90s from human host and P. falciparum with latter having fourfold higher affinity. A sequence analysis of the GA binding domain of Hsp90s from P. falciparum and human host showed a homologous substitution of K112 of hHsp90 to R98 in PfHsp90. In order to examine the effect of this substitution, if any, on the observed difference in GA binding abilities, we mutated R98 to K in PfHsp90. However, we did not find any difference in the binding ability of R98K PfHsp90 to GA, suggesting that this homologous substitution has minimal or no effect on drug protein interaction in vitro. However, in view of this phylogenetically conserved substitution, we cannot rule out its role in vivo. The chaperone function of Hsp90 is dependent on its ATPase activity which is susceptible to GA mediated inhibition. We next examined the extent of inhibition of GA on the ATPase activity of Hsp90s from P. falciparum and human host. Interestingly, we found the PfHsp90-ATPase activity to be three times more sensitive than hHsp90-ATPase activity to GA mediated inhibition suggesting that the malaria parasite, P. falciparum is likely to be more sensitive to GA when compared to human host. This result is in accordance with a recent study, which has shown that yeast expressing PfHsp90 in lieu of native yeast Hsp90 was more sensitive to GA than yeast expressing either yeast Hsp90 or human Hsp90.
Acetylation of Plasmodium falciparum Hsp90
Post-translational modification of Hsp90 such as acetylation has been shown to affect its binding with GA. We first examined whether, PfHsp90 can be acetylated. With the use of various purified Histone acetyl transferases (HATs) of human origin, we have shown PfHsp90 to undergo acetylation in vitro. We found that among different HATs (pCAF, Gcn5 and p300) used, only p300 was able to acetylate PfHsp90 suggesting a role for it in PfHsp90 in vivo acetylation as well. We next examined the in vivo acetylation status of PfHsp90 from parasite lysate. To enrich the acetylated fraction of PfHsp90, we have used Histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Immunoprecipitation of PfHsp90 followed by immunoblotting with an acetyl-lysine antibody confirmed that PfHsp90 undergoes acetylation in vivo. In order to identify the lysine residues which underwent acetylation we subjected the acetylation enriched fraction of PfHsp90 to in-gel trypsin digestion followed by mass spectrometry. Analysis of trypsin digested PfHsp90 from the parasites identified three sites of acetylation, one of which overlapped with PfHsp90 cochaperone (Aha1 and p23) binding residue, suggesting that acetylation could play a potential role in modulating PfHsp90 multi-chaperone complex assembly. Indeed, treatment of P. falciparum cultures with a HDAC-inhibitor resulted in partial dissociation of PfHsp90 complex as observed from size-exclusion chromatography. Adding to this observation, we also found that co-treatment of TSA and GA showed a synergistic and additive effect in inhibiting parasite growth in vitro. The above results suggest the possibility of using Hsp90 inhibitor in combination with HDAC inhibitor to arrest Plasmodium growth and development.
Clinically tested GA-analogue 17AAG inhibits Plasmodium growth in vitro and in vivo
The specificity of GA inside the cell has been a matter of debate since the discovery of its medicinal importance. In the past, Hsp90 has been implicated as a target of GA by carrying out immunoblotting of GA pull-down fraction with an anti-Hsp90 antibody. Crystal structure of GA with yeast Hsp90 has shown it to bind within the well conserved ATP-binding pocket of Hsp90. However, the specificity of GA inside the cell is still a conjecture. We have performed GA pull down assays from the parasite lysate followed by Coomassie Blue staining, which gave a single band corresponding to 86 kDa PfHsp90. The identity of PfHsp90 was further confirmed by immunoblotting with antibody specific to PfHsp90. This result indicates that inside the cells, inhibitory effect of GA is mediated by and large through its interaction with Hsp90. However, we cannot rule out the presence of other minor, less significant, interactors of GA.
Earlier work from our laboratory has shown that GA inhibits Plasmodium growth inside the infected erythrocytes. However, issues related to GA toxicity have excluded its development as a therapeutic. Nevertheless, interest in this class of molecule has led to the generation of a large number of less toxic derivatives of GA. One classical example is 17AAG which has gained clinical importance over the years and has entered in phase III trial. Intrigued by the clinical success of 17AAG, we were interested in determining its ability to modulate parasite growth. Indeed, 17AAG was able to inhibit parasite growth in a manner similar to that of GA. We further extended our study to parasites isolated from patient samples. Here too, we found 17AAG to be effective in inhibiting growth of the parasite. Finally, we examined the efficacy of 17AAG at a pre-clinical level using a mouse model of malaria. Using Peters’ four-day test we found 17AAG, to be effective in attenuating parasite growth and prolonging the survival of parasite infected mice (n=4, p=0.00692; n=10, p=0.001).
Clinical relevance of heat shock proteins of Plasmodium falciparum
A recent study using in vivo expression profiles of parasites derived from blood samples of infected patients has revealed previously unknown physiological diversity in the biology of malaria parasites. According to gene expression profiles, parasites were clustered into three different physiological states – starvation, glycolysis dependent active growth and environmental stress response. In order to examine the clinical relevance of molecular chaperones in malaria, we reanalyzed the previously published gene expression data of clinical parasites from 46 patients. Our analysis of this data showed that organellar chaperones were up-regulated upon starvation (cluster1) while cytosolic chaperones such as Hsp90 were up-regulated in active growth conditions (cluster2) indicating up-regulation of distinct group of Hsps in response to different environmental cues. Interestingly, Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, clustered in the same group. Further, some patients of cluster 3 (environmental stress response) showed higher expression of Hsp90 while others showed lower expression. In general, cluster 3 group of patients were heterogeneous in terms of expression of chaperones. Using non-negative matrix factorization (NMF), cluster 3 was sub-clustered into two groups 3a and 3b. Cluster 3b showed up-regulation of cytosolic chaperones similar to cluster 2 indicating these two clusters are inter-related. Most of the Hsp90 dependent pathways such as trafficking, signaling, anti apoptotic and pro-survival found to be most active in cluster 2 indicating the dependence of this group of parasites on Hsp90. The two main outcomes of our chaperone analysis are (1) the up-regulation of molecular chaperones in parasites are not a general response to hostile conditions as perceived previously, but is largely determined by the host factors and may differ from one host to another (2) the disease specific pathways may exist in natural condition by the up-regulation of specific chaperone and its interactors as a response to different host environment.
Clinical proteomics of human malarial parasites
Much of our understanding about the life cycle of parasites and importance of parasite proteins have been gleaned from the studies in laboratory strain or with the laboratory adapted clinical parasites. Although, these studies provide us first hand information about the functionality and the importance of these proteins, but they often fail to mimic the actual disease environment. In the patient, parasites are exposed to host factors such as hormones, metabolites, inflammatory mediators which can influence the expression of proteins and thus parasite biology. Further, instead of parasite exposure to 37°C temperature throughout the erythrocytic cycle in vitro, it is exposed to several rounds of febrile episodes inside human, which can also influence the parasite life cycle. Furthermore, clinical analysis is important to validate the presence and expression of drug targets in actual disease environment. Therefore, analysis of malaria parasite from clinical settings has become an important component in our laboratory and this thesis. Proteomic analysis of clinical samples has emerged as an important tool to understand the proteins dynamicity as response to disease environment. We have initiated clinical proteomic study of P. falciparum, the cause of most common and fatal malaria in humans and extended it further to the neglected malaria parasite P. vivax.
The study of P. vivax has largely been over-shadowed by the enormous attention devoted to P. falciparum. Notably, the drugs which have been discovered against P. falciparum are not as effective against P. vivax. Further several unique features of P. vivax such as dormant hyponozoites, reticulocyte host preference and formation of specialized caveolae vesicle complex structure distinguish its biology from P. falciparum and warrant concerted effort directed at this parasite. A major limitation in studying this parasite is the absence of a long-term culturing system. Therefore, research on this parasite requires samples obtained directly from patients. In spite of the inherent difficulty in obtaining such samples, this method provides us an opportunity to study this parasite in its real environment which has a huge effect on the expression as well as function of parasites and host proteins. Our current knowledge about the life cycle of this parasite has been gained from the recently published transcriptome study. Even though transcriptome analyses provide useful understanding at the level of gene expression, they do not reflect the active protein component of a cell. In other words, most of disease outcome is a result of interaction of the protein component with the environment. We therefore attempted to understand the protein component of this parasite in the disease environment to shed light on its pathogenicity. Despite facing several challenges in the way of proteomic analysis of this parasite such as availability of samples, low parasitemia, contamination of parasite proteins with abundant host proteins etc, we were able to identify 154 P. vivax proteins abundantly expressed in clinical environment using mass-spectrometry based approach. We found many proteins unique to this parasite along with known drug targets. This study is the first of its kind and could prove to be a very important step towards gaining insights into the physiology of this parasite.This study serves as a proof-of-principle method which in future is likely to help in identifying many more potential drug targets, vaccine candidates and diagnostic markers from clinically relevant samples as opposed to cultured samples.
Summary
Despite the importance of PfHsp90 in malaria biology, it has not been characterized in terms of its biochemical properties and its interaction with the inhibitor. In this study, we have successfully cloned, expressed, purified and characterized full length PfHsp90. We found that PfHsp90 exhibits a hyper-ATPase activity and is more sensitive to GA mediated inhibition as compared to human Hsp90. We have also shown that its sensitivity towards GA is dependent on its acetylation status as treatment of infected erythrocytes with HDAC inhibitors increases its sensitivity to GA. Using a pull-down assay, we have determined, unequivocally, that GA specifically binds to Hsp90. Most importantly, we have demonstrated that 17AAG, a clinically well-established inhibitor of Hsp90, inhibits parasite growth in a laboratory strain, field isolates and an in vivo mouse model of malaria. Overall, our biochemical characterization and drug interaction studies underscore the importance of PfHsp90 as a potent drug target and its inhibitors as a candidate drugs for the treatment of malaria, one of the deadly human infectious diseases.
Our efforts to understand the importance of molecular chaperones in parasites isolated directly from patient samples (clinical setting) has revealed conspicuous association of Hsps with previously defined parasite physiological states. In particular, parasites obtained from a specific group of patients exhibited heightened dependence on Hsp90-dependent pro-survival pathways, indicating an increased response to host stressors in this group of parasites. Thus, parasite encoded chaperones, in particular PfHsp90, play a major role in defining the pathogenesis of malaria.
A disease is an outcome of interaction between pathogens and its host, therefore it is important to study parasite in its real environment to understand disease pathogenesis. Our lab has previously reported the first ever proteomic analysis of P. falciparum from malaria patients. In this study, we have made an attempt to understand the unexplored biology of another important malaria parasite P. vivax. We have used a mass-spectrometry based approach to identify the protein content of this parasite. This technically challenging attempt has enabled us to identify many proteins. This study is an important step towards understanding the biology of this parasite in dearth of any information available on the proteins involved in this parasite’s pathogenicity
Functional Role Of Heat Shock Protein 90 From Plasmodium Falciparum
No full textMolecular chaperones have emerged in recent years as major players in many aspects of cell biology. Molecular chaperones are also known as heat shock proteins (HSPs) since many were originally discovered due to their
increased synthesis in response to heat shock. They were initially identified when
Drosophila salivary gland cells were exposed to a heat shock at 37°C for 30 min and then returned to their normal temperature of 25°C for recovery. A “puffing” of genes was found to have occurred in the chromosome of recovering cells, which was later shown to be accompanied by an increase in the synthesis of proteins with molecular masses of 70 and 26 kDa. These proteins were hence named “heat shock proteins”. The first identification of a function for HSPs was the
discovery in Escherichia coli that five proteins synthesized in response to heat
shock were involved in λ phage growth. The products of the groEL and groES genes were found to be essential for phage head assembly while the dnaK, dnaJ and grpE gene products were essential for λ phage replication. It was later shown that GroEL and GroES are part of a chaperonin system for protein folding in the prokaryotic cytosol while DnaK is a member of the Hsp70 family that works in conjunction with the DnaJ (Hsp40) co-chaperone and the nucleotide exchange factor GrpE to promote phage replication by dissociating the DnaB helicase from the phage-encoded P protein. Since then, a large number of other proteins
collectively referred to as HSPs have been discovered. However, heat shock is not the only signal that induces synthesis of heat shock proteins. Stress of any kind, such as nutrient deprivation, chemical treatment and oxidative stress among others causes increased production of HSPs and therefore, they are also known as stress proteins.
The term “molecular chaperone” was originally used to describe the function of nucleoplasmin, a Xenopus oocyte protein that promotes nucleosome assembly by binding tightly to histones and donating the bound histone to chromatin. However, since then, chaperones have been defined as “a family of
unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structure”. This view of
molecular chaperones, though undoubtedly correct, doesn’t capture the multifaceted roles they have since been discovered to play in cellular processes. In recent years, molecular chaperones have been shown to perform other functions in addition to the maintenance of protein homeostasis: translocation of proteins across organelle membranes, quality control in the endoplasmic reticulum, turnover of misfolded proteins as well as signal transduction. As a result, many chaperones are also essential under non-stress conditions and play crucial roles in cell growth and development, cell-cell communication and regulation of gene
expression.
Heat shock protein 90 (Hsp90) is one of the most abundant and highly conserved molecular chaperones in organisms ranging from bacteria to all branches of eukarya. It has been shown to be essential for cell viability in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila
melanogaster. Although the bacterial homolog HtpG is dispensable under normal conditions, it is important for cell survival during heat shock. In addition to its role as general chaperone in protein folding following stress, Hsp90 has a more
specialized role as a chaperone for several protein kinases and transcription factors. Many Hsp90 client proteins are signaling proteins involved in regulation of cell growth and survival. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance resulting in a key role for Hsp90 in these processes. Recent reports have also highlighted a role for Hsp90 in linking the expression of genetic and epigenetic variation in response to environmental stress with morphological development in Drosophila melanogaster and Arabidopsis thaliana. In Candida albicans, Hsp90 augments
the development of drug resistance, implicating a role for Hsp90 in the evolution
of infectious diseases.
The malarial parasite, Plasmodium falciparum, is the causative agent of
the most lethal form of human malaria. The parasite life cycle involves two hosts:
an invertebrate mosquito vector and a vertebrate human host. As the parasite
moves from the mosquito to the human body, it experiences an increase in temperature resulting in a severe heat shock. The mechanisms by which the parasite adapts to changes in temperature have not been deciphered. Our laboratory has been interested in investigating the role of heat shock proteins during acclimatization of the parasite to such temperature fluctuations. Heat shock proteins of the Hsp40, Hsp60, Hsp70 and Hsp90 families have been
characterized in the parasite and are being examined in our laboratory.
This thesis pertains to understanding the functional role of Plasmodium falciparum Hsp90 (PfHsp90) during adaptation of the parasite to fluctuations in environmental temperature. The parasite expresses a single gene for cytosolic Hsp90 on chromosome 7 (PlasmoDB accession no.: PF07_0029) coding for a protein of 745 amino acids with a pI of 4.94 and Mw of 86 kDa. Eukaryotic Hsp90
regulates several protein kinases and transcription factors involved in cell growth
and differentiation pathways resulting in a crucial role for Hsp90 in developmental
processes. A role for PfHsp90 in parasite development, therefore, seems likely. Indeed, PfHsp90 has previously been implicated in parasite development from
the ring stage to the trophozoite stage during the intra-erythrocytic cycle.
Pharmacological inhibition of PfHsp90 function using geldanamycin (GA), a
specific inhibitor of Hsp90 activity, abrogates stage progression. These
experiments suggest that PfHsp90 may play a critical role in parasite development. This is further substantiated by the fact that several pathogenic protozoan parasites such as Leishmania donovani, Trypanosoma cruzi,
Toxoplasma gondii and Eimeria tenella depend on Hsp90 function during different stages of their life cycles. It appears, therefore, that a principal role of Hsp90 in protozoan parasites may be the regulation of their developmental cycles. However, the precise functions of PfHsp90 during the intra-erythrocytic cycle of the malarial parasite are not clear.
In this study we have carried out a functional analysis of PfHsp90 in the
malarial parasite. We have examined the role of PfHsp90 in parasite development during repeated exposure to febrile temperatures. We have investigated its involvement in parasite development during a commonly used
synchronization protocol involving cyclical changes in temperature. We have examined the interaction of GA with the Hsp90 multi-chaperone complex from P. falciparum as well as the human host. Finally, we have carried out a systems level analysis of chaperone networks in the malarial parasite as well as its human host using an in silico approach. We have analyzed the protein-protein
interactions of PfHsp90 in the chaperone network and predicted putative cellular
processes likely to be regulated by parasite chaperones, particularly PfHsp90
Understanding the Biology of Heat Shock Protein 90 in Opportunistic Fungal Pathogens
Get PDFHeat shock protein 90 is one of the most abundant and evolutionary conserved class of molecular chaperones present throughout the biological kingdom. It is a specialist chaperone as its function extends beyond protein homeostasis. It is known to chaperone a specific set of proteins which lies at the interface of important cellular processes such as growth, signal transduction and developmental networks. It is believed that Hsp90 also functions as a stress responsive storehouse of genetic variation and thereby plays a key role in evolution. Another novel facet of Hsp90 was uncovered when the function of this chaperone was found to be important for pathogenesis of infectious disease-causing agents. Hsp90 was found to regulate stage transition in clinically important protozoan parasites such as Plasmodium, Giardia, Entamoeba and Leishmania.
Fungal pathogens cause substantial morbidity and mortality worldwide, however, the true impact of this group of pathogens on human health is not widely appreciated. Majority of fungal infections are often concealed under major comorbid conditions such as AIDS, cancer and other immunosuppressive conditions. Diagnosis and treatment of fungal infections is notoriously challenging due to emerging pathogens and drug resistance. Prospects of targeting Hsp90 to curb fungal pathogens has been explored in Candida albicans and Aspergillus fumigatus. Studies have revealed that Hsp90 governs one of the classical virulence traits of C. albicans, the yeast to hyphal transition. Additionally, the role of Hsp90 in potentiating antifungal resistance has been examined by genetic and inhibitor based studies. In this study, I have explored novel facets of Hsp90 in two clinically important fungal pathogens: Candida and Cryptococcus. Candida and Cryptococcus are the among the chief cause of death in cancer and AIDS patients. Furthermore, there is a surge in nosocomial fungal infections worldwide. I have investigated the hierarchy of Candida species implicated in invasive infections and found a clinical misdiagnosis of a multidrug resistant pathogen, Candida auris. Further, I have explored the role of Hsp90 in aiding resistance and thermotolerance of this emerging fungal pathogen.
Comparative analysis of Hsp90s of Candida and Cryptococcus
As mentioned previously, despite the great interest in examining the potential of Hsp90 to serve as a drug target to treat fungal infections, the basic biochemical properties of Hsp90s from fungal pathogens have not been studied previously. With this view, in Chapter 3, I have compared the biochemical properties of Hsp90 of these two fungal pathogens. Functionally, Hsp90 is an ATPase and the N-terminal domain of the protein harbours the nucleotide binding pocket. ATP hydrolysis is essential for its chaperoning ability. Bioinformatic analysis of the binding pocket of both Candida Hsp90 (CaHsp90) and Cryptococcus Hsp90 (CnHsp90) revealed subtle differences in the amino acid residues implicated in ATP binding, despite general conservation at the level of its primary structure. Many pharmacological inhibitors of Hsp90 such as geldanamycin and radicicol also bind to the nucleotide binding pocket and inhibit its activity. Therefore, we carried out in vitro ligand and inhibitor binding studies as well as ATPase activity studies using recombinant, bacterially expressed proteins obtained by cloning and purification. Both fungal Hsp90s i.e. CaHsp90 and CnHsp90 showed high affinity to ATP with a kd of 125.4 µM and 497.05 µM respectively. Further, the catalytic efficiencies of these Hsp90s was determined by measuring ATP hydrolysis rate using γ32 P ATP as a tracer. Catalytic efficiency of CaHsp90 was found to be 11.6 ×10-5 min-1µM-1 whereas CnHsp90 showed an efficiency of 6.39×10-5 min-1µM-1, indicating that CaHsp90 is a more active ATPase as compared to the Cryptococcus counterpart.
Pharmacological inhibitors of Hsp90 serve as a valuable molecular tool to understand protein function. We measured binding affinities of fungal Hsp90s to 17-AAG and we found that both CaHsp90 and CnHsp90 showed much higher affinity towards 17-AAG as compared to ATP with dissociation constants in range of 3-14 µM. Also, the ATPase activity of these fungal proteins was found to be more sensitive to pharmacological inhibition of Hsp90, indicating the possibility of drug development. Overall, we have carried out systematic biochemical analysis of Hsp90 of these two fungal pathogens.
Cellular functions of Hsp90 in Cryptococcus neoformans
In Chapter 4, I have investigated the role of Hsp90 in the pathogenesis and thermotolerance of C. neoformans, an environmental fungus that causes meningoencephalitis in humans. The genus Cryptococcus comprises over 37 species, most of which are environmental saprobes and non-pathogenic to humans. The pathogen has two unique virulence factors, melanised cell wall and a polysaccharide capsule which helps to combat host defences. However, many species of Cryptococcus such as C. podzolicus are equipped with these two pathogenicity armours but are not pathogenic to humans. Only two species which can grow at 37°C are human pathogens implicating the importance of thermotolerance for pathogenesis. The mechanism of growth at elevated temperature with respect to heat shock machinery has long been enigmatic. We found that thermotolerance of Cryptococcus critically depends on Hsp90 function as modest inhibition of Hsp90 function led to robust compromise in growth of the fungus at 37°C with little effect at 25°C. This observation also correlated with the finding that in vitro, 17-AAG showed a more potent inhibition of ATPase activity at 37°C. Interestingly, indirect immunofluorescence analysis using an antibody specific to CnHsp90 revealed cell surface localization via ER-Golgi classical secretory pathway. Furthermore, inhibition of Hsp90 function led to decrease in capsular volume and also improved the natural resistance of C. neoformans to cell wall targeting inhibitors echinocandins. Thus, Hsp90 dictates important virulence determinants of this pathogen.
Misdiagnosis of Candida auris
Numerous studies have linked Hsp90 to the pathogenic potential of fungi including yeast to hyphal transition, drug resistance and biofilm formation. However, these studies have been done in C. albicans because it is the leading fungal pathogen in the western countries. However, several epidemiological studies have reported the preponderance of non albicans Candida species in India. Therefore, we first sought to investigate the hierarchy of Candida species implicated in invasive infections. To address this, we collaborated with Manipal hospital, Bangalore, India. We found that C. tropicalis and C. parapsilosis were highly prevalent in this part of the world. During our study period, we noted the emergence of a new species called Candida haemulonii. Identification of the isolates was done by a commercial, automated system called Vitek2 by the hospital. Surprisingly, molecular studies in the lab revealed that these group of clinical isolates completely differ from the C. haemulonii wild type isolate. We further sequenced the whole genome of one of the clinical isolate Ci6684 and we found that it is Candida auris and was misdiagnosed to be C. haemulonii. In this study, we have developed a specific PCR to resolve the diagnostic dilemma between C. auris and C. haemulonii. Given that C. haemulonii genome is not sequenced, we have used the gene sequence for mating factor alpha as the candidate gene since it is known to be unique for each Candida species.
In 2016, C. auris outbreaks have been reported from many countries across the globe, including South Korea, India, Israel, South Africa, Japan, Canada, Germany, Colombia, Kenya, Spain, Norway, the United Kingdom, Pakistan and United States. We speculate that C. auris infections may also have occurred in other countries, however, the actual prevalence is underreported since the commercial automated systems used in clinics routinely misidentifies C. auris. This highlights the importance of early diagnosis of invasive candidiasis to initiate prompt treatment as delay in the administration of appropriate therapy increases mortality.
Genome of Candida auris
What makes misdiagnosis more alarming is the fact that these group of clinical isolates are not susceptible to the two frontline antifungals fluconazole and amphotericin B. To gain deeper insights into the biology of this misdiagnosed, multidrug resistant pathogen, we have generated the first draft genome of this pathogen which has been discussed in Chapter 6. The assembled draft genome of C. auris (clinical isolate Ci6684) consists of 99 scaffolds, 8358 protein coding genes, 189 tRNAs and 7 rRNAs. The draft genome size is 12.49 Mb, GC content 44.53% and 1.327 % Ns were estimated. Comparison of the genome of C. auris with other sequenced pathogenic Candida species by phylogeny and synteny analysis revealed that it has a highly divergent genome. Among the sequenced Candida species, C. auris was closest to C. lusitaniae as evident by substantial genomic collinearity seen in synteny dot plots and similarity in codon usage patterns. We further mined the genome to make an inventory of functional elements which can be potentially explain the pathogenicity mechanisms of C. auris. Enzyme classification analysis revealed that C. auris genome is enriched in hydrolases particularly, lipases, phospholipases and aspartyl proteinases. The activity of these enzymes is known to be high during invasive infections. Orthologs of genes implicated in biofilm formation, Rim101 transcriptional pathway and mitogen-activated protein kinase (MAPK) pathways were also conserved in C. auris. The draft genome sequence of C. auris will facilitate further studies to decipher the biology and virulence of C. auris.
Further, I have probed the antifungal resistant profile of clinical isolates of different Candida species including C. auris, C. tropicalis and C. haemulonii. Upon investigation of the prevalence of drug resistance in these species, we found that C. auris is currently the most resistant pathogen. Considering the conserved role of Hsp90 in antifungal resistance, we examined the effect of pharmacological inhibition of Hsp90 on drug resistant phenotype of these clinical isolates. Pharmacological inhibition of Hsp90 abrogated resistance in C. tropicalis and C. haemulonii. Surprisingly, Hsp90 inhibitors had no effect on fluconazole and amphotericin B resistance in C. auris. Genomic analysis revealed an enrichment of drug transport and metabolism pathways in C. auris. Further, we found that the pathogen is equipped with an arsenal of drug efflux pumps belonging to the ATP binding cassette (ABC) superfamily and major facilitator superfamily. Abundance of multidrug efflux pump genes encoding these drug transporters may explain the intrinsically low susceptibility of C. auris to antifungal drugs. Our analysis also predicted a multitude of zinc finger transcription factors which are known to modulate the expression of drug efflux pumps in response to antifungals. Furthermore, we also found that C. auris can grow at 42°C making it more thermotolerant as compared to other species. Interestingly, inhibition of Hsp90 led to complete abrogation of thermotolerance as evident by lack of growth at higher temperature. Further studies based on genetic compromise of Hsp90 function are needed to establish whether drug resistance in C. auris is Hsp90 independent or the effect seen is merely due to the activity of drug transporters.
Summary
Overall, in the thesis, we show that Hsp90s from two clinically important fungal pathogens differ in terms of their biochemical and cell biological properties. In Cryptococcus, Hsp90 was found to hone virulence traits including thermotolerance and capsulation. Interestingly, Hsp90 was also found to be cell surface localised and inhibition of Hsp90 pharmacologically improved anidulafungin tolerance at 37°C. We have also highlighted misdiagnosis of a multidrug resistant, emerging pathogen Candida auris. We have generated the first draft genome of this pathogen which facilitated the development of a molecular diagnostic method. The genome of C. auris Ci6684 is now the reference genome for this species. Analysis of the genome revealed the core virulence repertoire of C. auris which includes hydrolases, mannosyl transferases, adhesins and drug efflux pumps. Further, we found that although Hsp90 inhibition had no effect of on drug resistance of C. auris, however, Hsp90 inhibition led to abrogation of thermotolerance indicating the myriad ways by which it can be exploited by fungal pathogens
Metabolic Regulation of Gametocytogenesis in Malaria
No full textPlasmodium sp. is an obligate intracellular protozoan parasite causing malaria and is responsible for more than 500,000 human deaths annually. P. falciparum and P. vivax are the two major species causing malaria in humans. In the human host, the parasite exists in two forms; the asexual stages (merozoite, ring, trophozoite and schizont) and the sexual stage called gametocytes. The asexual intraerythrocytic cycle is responsible for the symptoms of malaria in the diseased host while gametocytes are essential for onward transmission of the parasite from a diseased host to a healthy host via the female Anopheles mosquito. A very small fraction of parasites participating in the asexual cycle exit the cycle to commit to form gametocytes. Since this is the only transmissible form of the parasite and important for development of transmission blocking interventions, it is critical to understand the factors responsible for commitment and development of mature gametocytes. In the current study we have used a combination of cell biology and bioinformatics along with global and targeted metabolomics to understand the cues and mechanisms of sexual stage induction in the malaria parasite.
Plasmodium being an intracellular pathogen closely shares its metabolic circuitry with host RBCs. In our study, we employed global and targeted metabolomics to investigate P. falciparum-induced host redox perturbation. We observed diminished GSH/GSSG ratio in parasitized RBC; indicative of redox imbalance in host cell. Further, we demonstrated that the parasite has a truncated TS pathway which leads to accumulation of redox active HCy in host RBCs and culture supernatant. This observation provides the mechanism behind the long-known occurrence of hyperhomocysteinemia in malaria patients. Additionally, we show that physiological concentration of HCy present in malaria patients is sufficient to induce sexual stage transition in the parasite to form gametocytes. Thus, we provide the functional significance of a disrupted metabolic pathway in the parasite. Using immunoblot and mass spectrometry we further observed a novel PTM called homocysteinylation in parasite histones which we hypothesize plays a role in epigenetic control of gametocytogenesis.
Further, we have studied the effect of redox imbalance in host RBCs on gametocytogenesis by examining (1) naturally occurring hemoglobinopathies like sickle cell anaemia and thalassemia. (2) G6PD-deficient RBCs; and (3) by creating redox stress using chemical intervention via 2-AAPA, an inhibitor of glutathione reductase. Our results show that in all the above host RBCs, redox imbalance triggers gametocytogenesis in parasites. Further, we found that plasma from haemoglobinopathic patients have elevated homocysteine levels and show concomitant increase in histone homocysteinylation of parasites growing in these RBCs, emphasising its potential role in triggering gametocytogenesis. Therefore, our observations indicate that hemoglobinopathic patients are protected from severe malaria but may work as silent reservoir of gametocytes facilitating transmission of malaria. Altogether our results implicate the potential of redox imbalance in triggering commitment and suggest the role of homocysteine and concomitant parasite histone homocysteinylation as a potential mechanism for autocrine/paracrine induction of gametocytogenesis. Apart from redox metabolites, our global metabolomics study revealed pipecolic acid to be uniquely present in parasite infected in vitro and in vivo samples. Thus, pipecolic acid may serve as a metabolite marker for malaria infections
Insights into life-cycle stage transition of Trichomonas vaginalis and advancement in its diagnosis
No full textTrichomonas vaginalis is a protozoan parasite and the causative agent of the most common non-viral, sexually transmitted disease (STD) in humans known as trichomoniasis. WHO estimates more than 270 million cases of trichomoniasis worldwide throughout the year. There is a lack in the understanding about its life cycle and mode of transmission. There are a few reports where non-sexual transmission is also observed, but the mechanism remains unclear. T. vaginalis is mostly prevalent in women in their reproductive age. Thus, the parasite must deal with changes in vaginal epithelium, cervical mucus, pH, redox potential, and overall modulation of the vaginal microbiome that occurs during the menstrual cycle. The survival and success of the parasite requires a robust and specialized machinery to tackle these stresses. The only drug of choice for trichomoniasis is metronidazole and drug-resistance are on the rise. There is also a lack of accurate diagnosis for the detection of T. vaginalis infection as the current diagnosis is mostly based on the clinical presentation of symptoms and is inaccurate.
The life cycle of Trichomonas vaginalis possess a trophozoite form which is transmitted sexually. In our study, we demonstrated the presence of Cyst-like structures (CLS) in the life cycle of T. vaginalis and characterized it under different stress conditions. This CLS form can survive in unfavourable conditions such as osmotic imbalance and presence of detergents and possess a thick cell membrane. This CLS stage can convert back to trophozoite form when favourable conditions return. The CLS form was observed to co-exist with trophozoite form in vivo in patient’s vaginal swab samples, indicating that CLS can also be infective. To understand the mechanism of formation of CLS, we have performed 2DGE and mass spectrometry based in-depth proteomics by using label free approach. We observed that the morphological transformation from trophozoite to CLS is coupled to less metabolic activity and alterations in proteins of adhesion and cytoskeletal reorganization. We also observed metronidazole resistance in CLS which is in range of the drug resistance observed in trophozoite forms of resistant strains.
In addition, we have biochemically characterized an important organelle present in Trichomonas known as hydrogenosome. Using mass spectrometry, we performed an exhaustive proteomics analysis to classify the identified proteins into their functional pathways. The study allows understanding of this ill-explored organelle present in an early branching eukaryote. We have developed a point-of-care diagnostic test for the detection of antibodies against the T. vaginalis infection in humans, which can be used in healthcare settings for accurate diagnosis.
Altogether, our study establishes a transmissible stage in the life cycle of T. vaginalis which can be transmitted non-sexually. In addition, the study highlights metabolic characterization, emergence of drug resistance and development of its point-of-care diagnostic tool for its infection
Insights Into Oxidative Folding Of Retinol Binding Protein In The Endoplasmic Reticulum : A Study In Isolated Microsomes
No full textThe central role played by the Endoplasmic Reticulum (ER) in the correct folding and assembly of secretary and membrane proteins cannot be overstated. As the first compartment in the secretary pathway, it is responsible for the synthesis, modification and targeting of proteins to their proper destinations within the secretary pathway and the extracellular space. Protein folding in this specialized compartment is dynamic and involves a host of molecular chaperones and folding catalysts. Once inside the ER lumen, proteins fold into their native conformation and undergo a multitude of post-translational modifications, including N-linked glycosylation and disulfide oxidation. The proper conformational maturation of nascent proteins that traverse the secretary pathway is both aided and monitored by a complex process termed ER quality control. A variety of quality control mechanisms that rely on the chaperone systems operate in the ER. These act in close concert with the molecular machinery involved in degradation of non-native proteins to maintain homeostasis. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. As a general rule, only those proteins that have successfully completed their folding and passed a stringent selection process are allowed to exit the ER on their way to their final destinations. The importance of the normal functioning of the ER is underlined by the fact that disruption in protein folding, resulting in ER stress, has now been identified as the biochemical basis of many ER storage diseases including Diabetes mellitus, Endocrinopathies and Hemophilia A.
Processing events occurring inside the ER lumen are known to influence the efficiency of protein secretion. Vastly different rates of exocytose observed among secretary proteins have been found to correlate with the rate of exit from the ER. One such example is the interesting secretion property exhibited by Retinol Binding Protein (RBP)
The principal carrier of retinol (Vitamin A) in plasma. RBP is a single domain protein consisting of three intramolecular disulfide bonds and helps transport retinol from the liver stores to the various target tissues in the body. Availability of its ligand, retinol, while not affecting its synthesis, is known to be the major factor in regulating RBP secretion from the liver. In the absence of retinol, apo-RBP has been shown to be retained in the ER by a hitherto unclear mechanism.
Like most other secretary proteins, RBP is co-translationally targeted to the ER lumen, where it undergoes disulfide oxidation as the only modification. It has been shown to form a complex with another secretary protein, Transthyretin (TTR) in the ER and this complex formation is thought to prevent premature glomerular filtration of the otherwise small RBP with its bound retinol. Despite attaining a mature conformation, apo-RBP is not secreted and awaits conversion to its ligand-bound, holo form in order to exit the ER. It is widely believed that ligand binding may relieve this retention of RBP from the ER quality control machinery. However the precise mechanisms that mediate and regulate RBP folding, ligand binding, TTR assembly and secretion are not clearly understood. Though the folding and secretion properties of RBP have been described in HepG2 cells, its interactions with the ER resident chaperones have not been addressed. Apart from being an important cell biological question, the study of RBP assumes a lot of significance with its recent emergence as a key player in the pathogenesis of type 2 diabetes mellitus. It has been proposed that lowering of serum RBP levels could be a new strategy for treating type 2 diabetes mellitus.
The present study was undertaken with the intention of analyzing the oxidative folding of RBP in the ER more closely. A systematic approach aimed at understanding the early events associated with folding and maturation of RBP, with particular emphasis on the role of ER-resident chaperones and the quality control machinery, is likely to provide interesting insights into the mechanisms involved in its ligand dependent secretion.
Reconstitution of RBP biogenesis in a cell free system.
The folding of RBP in cells is extremely quick with rapid oxidation kinetics. This makes it difficult to systematically analyze the early folding events in cultured cells. It was necessary to make use of a simplified system that would faithfully recapitulate the folding process in the ER. Therefore, a cell free translation system consisting of rabbit reticulocyte lysate and canine pancreatic microcosms as a source of ER-derived membranes was developed. This system affords the advantage of easy manipulation while still preserving the overall environment that prevails in the ER of intact cells. Extensive biochemical and functional characterization of the isolated microcosms was carried out and in vitro translation and microsomal translocation of RBP was established. Though initially confined to studies on membrane insertion and core glycosylate, the cell free system supplemented with microcosms has subsequently been used to analyze folding and assembly of a number of secretary and membrane proteins. A similar strategy has been adopted in the present study of RBP folding and maturation.
Oxidative folding of RBP in isolated microcosms: Delineation of its disulfide oxidation pathway
Using glutathione (GSSG) as the oxidant, co- and posttranslational disulfide oxidation of RBP was carried out in isolated microcosms. The ability to manipulate the redox status of this cell free system has helped to considerably slow down the oxidative folding of RBP so that a more careful analysis of the folding process could be performed. RBP was found to undergo oxidative folding with a t1/2 of 30 minutes and folding proceeded through at least one disulfide-bonded intermediate. Non-reducing SDS PAGE was used to resolve the folding intermediates. The pattern of oxidation was in good agreement with that reported earlier in HepG2 cells. No significant effect of retinol was observed on either the folding kinetics or the pattern of disulfide oxidation of RBP in isolated microsomes.A DTT sensitivity assay, used to probe the conformational maturity of folding RBP, revealed that RBP was capable of maturing into a DTT-resistant conformation in isolated microsomes.
With the aid of disulfide mutants, the probable disulfide oxidation pathway of RBP in the ER has been determined. Single and double disulfide mutants of RBP were generated by site-directed mutagenesis and their posttranslational oxidation patterns were analyzed and compared with that of the wild type protein. Based on the results obtained, it was clear that the folding intermediate was made up of one of the two big disulfide loops and that the presence of both these loops was essential for RBP to fold into a fully oxidized, compact form. It has not been possible to determine the contribution of the third, smallest disulfide loop to the oxidative folding of RBP.
Molecular events associated with the early oxidative folding of RBP
To gain insights into the possible role of ER chaperones in the oxidative folding of RBP, the oligomeric state of folding RBP was analyzed by velocity sedimentation and chemical crosslinking assays. Velocity sedimentation analysis revealed that the reduced form of RBP was present in a large complex of size >100 S20,W. Upon disulfide oxidation, it readily dissociated from the complex and assumed a monomeric state. This was evident even during co-translational oxidation which suggested that RBP transiently associated with the large complex during its oxidative folding. Dynamic nature of this complex indicated that this could be a folding complex containing the chaperone machinery of the ER. These results were also supported by crosslinking analysis performed in unbroken microsomes using the homo-bifunctional crosslinker, DSP. The early folding forms of RBP could be crosslinked to a large complex while upon disulfide oxidation, RBP matured to its monomeric form and was no longer crosslinkable. Sedimentation and crosslinking analyses of the RBP disulfide mutants revealed that while the double disulfide mutant remained irreversibly associated with the large complex, the single mutants were released upon acquiring one of the two big disulfide loops. This suggested that despite the lack of one of the two major disulfides, these mutants were considered ‘folded’ by the quality control machinery in the ER while the double mutant probably resembled a molten globule state and was therefore considered ‘unfolded’ and irreversibly retained. Results from crosslinking analysis in microsomes not engaged in active translation suggested that chaperones of the ER were organized in a complex constitutively thereby lending support to the concept of ER-matrix, a large network of luminal proteins consisting of ER chaperones and accessory factors. Given this scenario, it is not unlikely that newly synthesized protein substrates transiently associate with this large pre-existing complex of chaperones and dissociate during late stages of their maturation.
Conclusion
In all, this study provides significant insights into some of the early events associated with the oxidative folding of RBP in the ER. The delineation of the disulfide oxidation pathway of RBP has been possible. The results obtained from this study suggest that RBP probably dissociates from the quality control quite early during its folding process and this step in its maturation might not be influenced by retinol. The stimulus for its ligant dependent secretion is likely to operate at a later stage of its sojourn in the ER, possibly consequent to positive cues from accessory binding factors such as TTR. Lastly, Perservation of the ER microenvironment in isolated microsomes, as evidenced from this study, augurs well for the use of this system to analyze mechanisms underlying folding, maturation, secretion and/or retention of secretory proteins
Understanding the Heat Shock Response Pathway in Plasmodium Falciparum and Identification of a Novel Exported Heat Shock Protein
Get PDFInfections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose.
During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock.
The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway.
Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress.
Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis
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