Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently... more Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based ‘omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our system...
Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as ... more Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer’s disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administere...
The hsp90 chaperones govern the function of essential client proteins critical to normal cell fun... more The hsp90 chaperones govern the function of essential client proteins critical to normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain (NTD) and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP binding pocket of the NTD have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and β, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP binding pockets of the four paralogs are flanked by three side pockets, termed Sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to Sites 1 and 2 have resulted in the development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of Site 3 are less clear. Previous work identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here, we use NECA and its derivatives to probe the properties of Site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into Site 3 of Grp94 compared to their parent compound and that selectivity is due to paralogspecific differences in ligand pose and ligandinduced conformational strain in the protein. These studies provide a structural basis for Grp94selective inhibition using Site 3.
Cellular transformation is accompanied by extensive rewiring of many biological processes leading... more Cellular transformation is accompanied by extensive rewiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress respo...
A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is ... more A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is described. We demonstrate the use of CuTC as a soluble, non-basic catalyst in the coupling of aryl iodides and aryl thiols in the synthesis of synthetically advanced diaryl sulfides. This method allows for the successful coupling of challenging substrates including -substituted and heteroaryl iodides and thiols. Additionally, most of the aryl iodide substrates used here contain the privileged piperazine scaffold bound to a pyrimidine, pyridine, or phenyl ring and thus this method allows for the elaboration of complex piperazine scaffolds into molecules of biological interest. The method described here enables the incorporation of late-stage structural diversity into diaryl sulfides containing the piperazine ring, thus enhancing the number and nature of derivatives available for SAR investigation.
The chaperome is a large and diverse protein machinery composed of chaperone proteins and a varie... more The chaperome is a large and diverse protein machinery composed of chaperone proteins and a variety of helpers, such as the co-chaperones, folding enzymes, and scaffolding and adapter proteins. Heat shock protein 90s and 70s (HSP90s and HSP70s), the most abundant chaperome members in human cells, are also the most complex. As we have learned to appreciate, their functions are context dependent and manifested through a variety of conformations that each recruit a subset of co-chaperone, scaffolding, and folding proteins and which are further diversified by the posttranslational modifications each carry, making their study through classic genetic and biochemical techniques quite a challenge. Chemical biology tools and techniques have been developed over the years to help decipher the complexities of the HSPs and this review provides an overview of such efforts with focus on HSP90 and HSP70. (A) Ribbon representation of the apo full-length monomer HtpG (Escherichia coli HSP90, PDB: 2IOQ) and the reported mode of interaction for select HSP90 inhibitors. The chemical structure of each ligand is depicted on the right side. (B) Geldanamycin (GM)-derived HSP90 chemical probes. (C) Other HSP90 chemical probes.
Background: Hsp70, a molecular chaperone responsible, in part, for the folding of nascent peptide... more Background: Hsp70, a molecular chaperone responsible, in part, for the folding of nascent peptides following translation, has been implicated as a survival factor and a poor prognostic marker in cancer cells. These pro-cancer mechanisms originate in the ability of Hsp70 to preserve and maintain oncogenic and transformative proteins responsible for the cancer phenotype. Hsp70 is a stress response protein such that expression increases under proteomic/proteotoxic stress events. This increased expression is also evident in cancer cells, an environment under proteomic stress brought on by transformation, and is known to be protective against programmed cell death. Hypothesis: We predict that by interrupting the chaperoning capacity of Hsp70 through allosteric inhibition, we can destabilize oncogenic proteins dependent on Hsp70 for structure and function. Additionally, as Hsp70 is a survival factor, we believe that a loss of Hsp70 activity will result in cancer specific cell death both i...
Background: Heat shock protein 70 family members play an important role in cancer. They are up-re... more Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its functio...
International journal for numerical methods in biomedical engineering, 2014
Biomedical flow computations in patient-specific geometries require integrating image acquisition... more Biomedical flow computations in patient-specific geometries require integrating image acquisition and processing with fluid flow solvers. Typically, image-based modeling processes involve several steps, such as image segmentation, surface mesh generation, volumetric flow mesh generation, and finally computational simulation. These steps are performed separately, often using separate pieces of software, and each step requires considerable expertise and investment of time on the part of the user. In this paper an alternative framework is presented in which the entire image-based modeling process is performed on a Cartesian domain where the image is embedded within the domain as an implicit surface. Thus the framework circumvents the need for generating surface meshes to fit complex geometries and subsequent creation of body-fitted flow meshes. Cartesian mesh pruning, local mesh refinement, and massive parallelization provide computational efficiency; the image-to-computation techniques adopted are chosen to be suitable for distributed memory architectures. The complete framework is demonstrated with flow calculations computed in two 3D image reconstructions of geometrically dissimilar intracranial aneurysms. The flow calculations are performed on multiprocessor computer architectures and are compared against calculations performed with a standard multi-step route.
Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Ca... more Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. T he heat shock protein 70 family members (Hsp70s) are important cancer chaperones. They are abundantly expressed in malignant tumors of various origins, and their expression correlates with increased cell proliferation, poor differentiation, metastases, resistance to therapies, and poor therapeutic outcome in human cancers. 1−9 A variety of mechanisms have been assigned to account for the observed reliance of cancer cells on Hsp70s, including inhibition of apoptosis, induction of autophagy, control of senescence, and regulation of the stability of onco-proteins. 1−9 Much of our knowledge on Hsp70 function in cancer is derived from genetic knockdown approaches. 6 Performing knock-down studies can however be challenging as Hsp70 is a family of at least 8 isoforms, some of interchangeable functions, a significant number with long half-lives and high constitutive expression. 6,7 Genetic studies also treat Hsp70 as a monolithic entity and are unable to tackle the acknowledged contribution of epigenetics to the activity of these proteins. Further, due to feedback synthesis of one Hsp70 member after the knock-down of another, such studies often lead to no observable phenotypes. 6−9 Cellular manipulations that are often conducted to investigate the function of a protein and its potential interactors, i.e., by transfection of mutants, tagged proteins, or overexpression systems, need also caution as they may
Studies using simulated calcifications can be performed to measure the effect of different imagin... more Studies using simulated calcifications can be performed to measure the effect of different imaging factors on calcification detection in digital mammography. The simulated calcifications must be inserted into clinical images with realistic contrast and sharpness. MoCa is a program which modifies the contrast and sharpness of simulated calcification clusters extracted from images of mastectomy specimens acquired on a digital specimen cabinet at high magnification for insertion into clinical mammography images. This work determines whether the use of MoCa results in simulated calcifications with the correct contrast and sharpness. Aluminium foils (thickness 0.1-0.4 mm) and 1.60 µm thick gold discs (diameter 0.13-0.8 mm) on 0.5 mm aluminium were imaged with a range of thicknesses of polymethyl methacrylate (PMMA) using an amorphous selenium direct digital (DR) system and a powder phosphor computed radiography (CR) system (real images). Simulated images of the tests objects were also generated using MoCa. The contrast of the aluminium squares and the degradation of the contrast of the gold discs as a function of disc diameter were compared in the real and simulated images. The average ratios of the simulated-to-real aluminium contrasts over all aluminium and PMMA thicknesses were 1.03 ± 0.04 (two standard errors in the mean) and 0.99 ± 0.03 for the DR and CR systems, respectively. The ratio of the simulated-to-real degradations of contrast averaged over all disc diameters and PMMA thicknesses were 1.007 ± 0.008 and 1.002 ± 0.013 for DR and CR, respectively. The use of MoCa was accurate within the experimental errors.
Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for ... more Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-termimal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-Sfarnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng-Prusoff method, the Ki values were determined from the IC 50 values. Analog 1a has a K IC of 1.4 ± 0.2 μM and a K IU of 4.8 ± 0.5 μM while 1b has a K IC of 0.5 ± 0.07 μM and a K IU of 1.9 ± 0.2 μM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells.
Biochemical and Biophysical Research Communications, 2012
Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein me... more Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and S. cerevisae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast afactor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the afactor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition and that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. These data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites.
Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently... more Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based ‘omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our system...
Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as ... more Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer’s disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administere...
The hsp90 chaperones govern the function of essential client proteins critical to normal cell fun... more The hsp90 chaperones govern the function of essential client proteins critical to normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain (NTD) and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP binding pocket of the NTD have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and β, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP binding pockets of the four paralogs are flanked by three side pockets, termed Sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to Sites 1 and 2 have resulted in the development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of Site 3 are less clear. Previous work identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here, we use NECA and its derivatives to probe the properties of Site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into Site 3 of Grp94 compared to their parent compound and that selectivity is due to paralogspecific differences in ligand pose and ligandinduced conformational strain in the protein. These studies provide a structural basis for Grp94selective inhibition using Site 3.
Cellular transformation is accompanied by extensive rewiring of many biological processes leading... more Cellular transformation is accompanied by extensive rewiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress respo...
A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is ... more A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is described. We demonstrate the use of CuTC as a soluble, non-basic catalyst in the coupling of aryl iodides and aryl thiols in the synthesis of synthetically advanced diaryl sulfides. This method allows for the successful coupling of challenging substrates including -substituted and heteroaryl iodides and thiols. Additionally, most of the aryl iodide substrates used here contain the privileged piperazine scaffold bound to a pyrimidine, pyridine, or phenyl ring and thus this method allows for the elaboration of complex piperazine scaffolds into molecules of biological interest. The method described here enables the incorporation of late-stage structural diversity into diaryl sulfides containing the piperazine ring, thus enhancing the number and nature of derivatives available for SAR investigation.
The chaperome is a large and diverse protein machinery composed of chaperone proteins and a varie... more The chaperome is a large and diverse protein machinery composed of chaperone proteins and a variety of helpers, such as the co-chaperones, folding enzymes, and scaffolding and adapter proteins. Heat shock protein 90s and 70s (HSP90s and HSP70s), the most abundant chaperome members in human cells, are also the most complex. As we have learned to appreciate, their functions are context dependent and manifested through a variety of conformations that each recruit a subset of co-chaperone, scaffolding, and folding proteins and which are further diversified by the posttranslational modifications each carry, making their study through classic genetic and biochemical techniques quite a challenge. Chemical biology tools and techniques have been developed over the years to help decipher the complexities of the HSPs and this review provides an overview of such efforts with focus on HSP90 and HSP70. (A) Ribbon representation of the apo full-length monomer HtpG (Escherichia coli HSP90, PDB: 2IOQ) and the reported mode of interaction for select HSP90 inhibitors. The chemical structure of each ligand is depicted on the right side. (B) Geldanamycin (GM)-derived HSP90 chemical probes. (C) Other HSP90 chemical probes.
Background: Hsp70, a molecular chaperone responsible, in part, for the folding of nascent peptide... more Background: Hsp70, a molecular chaperone responsible, in part, for the folding of nascent peptides following translation, has been implicated as a survival factor and a poor prognostic marker in cancer cells. These pro-cancer mechanisms originate in the ability of Hsp70 to preserve and maintain oncogenic and transformative proteins responsible for the cancer phenotype. Hsp70 is a stress response protein such that expression increases under proteomic/proteotoxic stress events. This increased expression is also evident in cancer cells, an environment under proteomic stress brought on by transformation, and is known to be protective against programmed cell death. Hypothesis: We predict that by interrupting the chaperoning capacity of Hsp70 through allosteric inhibition, we can destabilize oncogenic proteins dependent on Hsp70 for structure and function. Additionally, as Hsp70 is a survival factor, we believe that a loss of Hsp70 activity will result in cancer specific cell death both i...
Background: Heat shock protein 70 family members play an important role in cancer. They are up-re... more Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its functio...
International journal for numerical methods in biomedical engineering, 2014
Biomedical flow computations in patient-specific geometries require integrating image acquisition... more Biomedical flow computations in patient-specific geometries require integrating image acquisition and processing with fluid flow solvers. Typically, image-based modeling processes involve several steps, such as image segmentation, surface mesh generation, volumetric flow mesh generation, and finally computational simulation. These steps are performed separately, often using separate pieces of software, and each step requires considerable expertise and investment of time on the part of the user. In this paper an alternative framework is presented in which the entire image-based modeling process is performed on a Cartesian domain where the image is embedded within the domain as an implicit surface. Thus the framework circumvents the need for generating surface meshes to fit complex geometries and subsequent creation of body-fitted flow meshes. Cartesian mesh pruning, local mesh refinement, and massive parallelization provide computational efficiency; the image-to-computation techniques adopted are chosen to be suitable for distributed memory architectures. The complete framework is demonstrated with flow calculations computed in two 3D image reconstructions of geometrically dissimilar intracranial aneurysms. The flow calculations are performed on multiprocessor computer architectures and are compared against calculations performed with a standard multi-step route.
Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Ca... more Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. T he heat shock protein 70 family members (Hsp70s) are important cancer chaperones. They are abundantly expressed in malignant tumors of various origins, and their expression correlates with increased cell proliferation, poor differentiation, metastases, resistance to therapies, and poor therapeutic outcome in human cancers. 1−9 A variety of mechanisms have been assigned to account for the observed reliance of cancer cells on Hsp70s, including inhibition of apoptosis, induction of autophagy, control of senescence, and regulation of the stability of onco-proteins. 1−9 Much of our knowledge on Hsp70 function in cancer is derived from genetic knockdown approaches. 6 Performing knock-down studies can however be challenging as Hsp70 is a family of at least 8 isoforms, some of interchangeable functions, a significant number with long half-lives and high constitutive expression. 6,7 Genetic studies also treat Hsp70 as a monolithic entity and are unable to tackle the acknowledged contribution of epigenetics to the activity of these proteins. Further, due to feedback synthesis of one Hsp70 member after the knock-down of another, such studies often lead to no observable phenotypes. 6−9 Cellular manipulations that are often conducted to investigate the function of a protein and its potential interactors, i.e., by transfection of mutants, tagged proteins, or overexpression systems, need also caution as they may
Studies using simulated calcifications can be performed to measure the effect of different imagin... more Studies using simulated calcifications can be performed to measure the effect of different imaging factors on calcification detection in digital mammography. The simulated calcifications must be inserted into clinical images with realistic contrast and sharpness. MoCa is a program which modifies the contrast and sharpness of simulated calcification clusters extracted from images of mastectomy specimens acquired on a digital specimen cabinet at high magnification for insertion into clinical mammography images. This work determines whether the use of MoCa results in simulated calcifications with the correct contrast and sharpness. Aluminium foils (thickness 0.1-0.4 mm) and 1.60 µm thick gold discs (diameter 0.13-0.8 mm) on 0.5 mm aluminium were imaged with a range of thicknesses of polymethyl methacrylate (PMMA) using an amorphous selenium direct digital (DR) system and a powder phosphor computed radiography (CR) system (real images). Simulated images of the tests objects were also generated using MoCa. The contrast of the aluminium squares and the degradation of the contrast of the gold discs as a function of disc diameter were compared in the real and simulated images. The average ratios of the simulated-to-real aluminium contrasts over all aluminium and PMMA thicknesses were 1.03 ± 0.04 (two standard errors in the mean) and 0.99 ± 0.03 for the DR and CR systems, respectively. The ratio of the simulated-to-real degradations of contrast averaged over all disc diameters and PMMA thicknesses were 1.007 ± 0.008 and 1.002 ± 0.013 for DR and CR, respectively. The use of MoCa was accurate within the experimental errors.
Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for ... more Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-termimal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-Sfarnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng-Prusoff method, the Ki values were determined from the IC 50 values. Analog 1a has a K IC of 1.4 ± 0.2 μM and a K IU of 4.8 ± 0.5 μM while 1b has a K IC of 0.5 ± 0.07 μM and a K IU of 1.9 ± 0.2 μM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells.
Biochemical and Biophysical Research Communications, 2012
Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein me... more Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and S. cerevisae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast afactor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the afactor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition and that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. These data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites.
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Papers by Liza Shrestha