Barak Rotblat | Ben Gurion University of the Negev (original) (raw)
Papers by Barak Rotblat
Cell, 2013
Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that e... more Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:
Oncotarget, Jan 30, 2014
The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, a... more The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additional...
The spatial organization of K-Ras proteins into nanoclusters on the plasma membrane is essential ... more The spatial organization of K-Ras proteins into nanoclusters on the plasma membrane is essential for high-fidelity signal transduction. The mechanism underlying K-Ras nanocluster- ing is unknown. We show here that K-Ras.GTP recruits Galectin-3 (Gal-3) from the cytosol to the plasma membrane where it becomes an integral nanocluster component. Importantly, we show that the cytosolic level of Gal-3 determines the magnitude
The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for ... more The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters.
Oncotarget, 2013
Cells mount a transcriptional anti-oxidative stress (AOS) response program to scavenge reactive o... more Cells mount a transcriptional anti-oxidative stress (AOS) response program to scavenge reactive oxygen species (ROS) that arise from chemical, physical, and metabolic challenges. This protective program has been shown to reduce carcinogenesis triggered by chemical and physical insults. However, it is also hijacked by established cancers to thrive and proliferate within the hostile tumor microenvironment and to gain resistance against chemo- and radiotherapies. Therefore, targeting the AOS response proteins that are exploited by cancer cells is an attractive therapeutic strategy. In order to identify the AOS genes that are suspected to support cancer progression and resistance, we analyzed the expression patterns of 285 genes annotated for being involved in oxidative stress in 994 tumors and 353 normal tissues. Thereby we identified a signature of 116 genes that are highly overexpressed in multiple carcinomas while being only minimally expressed in normal tissues. To establish which ...
Cell death & disease, 2014
Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is ... more Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine--a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are...
Oncotarget, 2012
The transcription factor nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF... more The transcription factor nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, is a master regulator of the anti-oxidative stress response and positively controls the expression of a battery of anti-oxidative stress response proteins and enzymes implicated in detoxification and glutathione generation. Although its detoxifying activity is important in cancer prevention, it has recently been shown that cancer cells also exploit its protective functions to thrive and resist chemotherapy. NRF2 was also shown to the pentose phosphate pathway and glutaminolysis, which promotes purine synthesis for supporting rapid proliferation and glutathione for providing anti-oxidative stress protection. Evidence obtained from cancer patients and cell lines suggest that NRF2 is highly active in a variety of human cancers and is associated with aggressiveness. p53 is a tumor suppressor that also promotes an anti-oxidative stress metabolic program and glutaminolysis. Here we will dis...
The Plant journal : for cell and molecular biology, 2002
Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene bio... more Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-1-4-endoxylanase activity of the EIX protein further demonstrating that ...
Spatiotemporal modulation of Ras signaling from different intracellular compartments requires mec... more Spatiotemporal modulation of Ras signaling from different intracellular compartments requires mechanisms allowing Ras and its signals to navigate across cells. Here, we describe one mechanism by which clusters of palmitoylated H-Ras and N-Ras isoforms but not nonpalmitoylated K-Ras diffuse through the cytoplasm, independently of ATP, on fast, randomly moving, small cytosolic nanoparticles (''raso- somes''). Rasosomes forced to diffuse out of
Molecular and cellular biology, 2004
The microlocalization of Ras proteins to different microdomains of the plasma membrane is critica... more The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segreg...
The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, a... more The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides.
Methods in Enzymology, 2008
Chronic activation of Ras proteins by mutational activation or by growth factor stimulation is a ... more Chronic activation of Ras proteins by mutational activation or by growth factor stimulation is a common occurrence in many human cancers and was shown to induce and be required for tumor growth. Even if additional genetic defects are present, "correction" of the Ras defect has been shown to reverse Ras-dependent tumorigenesis. One way to block Ras protein activity is by interfering with their spatiotemporal localization in cellular membranes or in membrane microdomains, a prerequisite for Ras signaling and biological activity. Detailed reports describe the use of this method in studies employing farnesylthiosalicylic acid (FTS, Salirasib), a Ras farnesylcysteine mimetic, which selectively disrupts the association of chronically active Ras proteins with the plasma membrane. FTS competes with Ras for binding to Ras-escort proteins, which possess putative farnesyl-binding domains and interact only with the activated form of Ras proteins, thereby promoting Ras nanoclusterization in the plasma membrane and robust signals. This chapter presents three-dimensional time-lapse images that track the FTS-induced inhibition of membrane-activated Ras in live cells on a real-time scale. It also describes a mechanistic model that explains FTS selectivity toward activated Ras. Selective blocking of activated Ras proteins results in the inhibition of Ras transformation in vitro and in animal models, with no accompanying toxicity. Phase I clinical trials have demonstrated a safe profile for oral FTS, with minimal side effects and promising activity in hematological malignancies. Salirasib is currently undergoing trials in patients with pancreatic cancer and with nonsmall cell lung cancer, with or without identified K-Ras mutations. The findings might indicate whether with the disruption of the spatiotemporal localization of oncogenic Ras proteins and the targeting of prenyl-binding domains by anticancer drugs is worth developing as a means of cancer treatment.
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2014
Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment,... more Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment, including hypoxia, nutrient deprivation, and oxidative or genotoxic stress. Tumor cells must evolve adaptive mechanisms to survive these conditions to ultimately drive tumor progression. Tight control of mRNA translation is critical for this response and the adaptation of tumor cells to such stress forms. This proceeds though a translational reprogramming process which restrains overall translation activity to preserve energy and nutrients, but which also stimulates the selective synthesis of major stress adaptor proteins. Here we present the different regulatory signaling pathways which coordinate mRNA translation in the response to different stress forms, including those regulating eIF2α, mTORC1 and eEF2K, and we explain how tumor cells hijack these pathways for survival under stress. Finally, mechanisms for selective mRNA translation under stress, including the utilization of upstream open reading frames (uORFs) and internal ribosome entry sites (IRESes) are discussed in the context of cell stress. This article is part of a Special Issue entitled: Translation and Cancer.
Oncogene, 2014
Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative d... more Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration.
Traffic, 2006
Ras signaling to its downstream effectors appears to include combinations of extracellular-signal... more Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with 'rasosomes', randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function.
Cell, 2013
Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that e... more Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:
Oncotarget, Jan 30, 2014
The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, a... more The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additional...
The spatial organization of K-Ras proteins into nanoclusters on the plasma membrane is essential ... more The spatial organization of K-Ras proteins into nanoclusters on the plasma membrane is essential for high-fidelity signal transduction. The mechanism underlying K-Ras nanocluster- ing is unknown. We show here that K-Ras.GTP recruits Galectin-3 (Gal-3) from the cytosol to the plasma membrane where it becomes an integral nanocluster component. Importantly, we show that the cytosolic level of Gal-3 determines the magnitude
The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for ... more The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters.
Oncotarget, 2013
Cells mount a transcriptional anti-oxidative stress (AOS) response program to scavenge reactive o... more Cells mount a transcriptional anti-oxidative stress (AOS) response program to scavenge reactive oxygen species (ROS) that arise from chemical, physical, and metabolic challenges. This protective program has been shown to reduce carcinogenesis triggered by chemical and physical insults. However, it is also hijacked by established cancers to thrive and proliferate within the hostile tumor microenvironment and to gain resistance against chemo- and radiotherapies. Therefore, targeting the AOS response proteins that are exploited by cancer cells is an attractive therapeutic strategy. In order to identify the AOS genes that are suspected to support cancer progression and resistance, we analyzed the expression patterns of 285 genes annotated for being involved in oxidative stress in 994 tumors and 353 normal tissues. Thereby we identified a signature of 116 genes that are highly overexpressed in multiple carcinomas while being only minimally expressed in normal tissues. To establish which ...
Cell death & disease, 2014
Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is ... more Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine--a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are...
Oncotarget, 2012
The transcription factor nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF... more The transcription factor nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, is a master regulator of the anti-oxidative stress response and positively controls the expression of a battery of anti-oxidative stress response proteins and enzymes implicated in detoxification and glutathione generation. Although its detoxifying activity is important in cancer prevention, it has recently been shown that cancer cells also exploit its protective functions to thrive and resist chemotherapy. NRF2 was also shown to the pentose phosphate pathway and glutaminolysis, which promotes purine synthesis for supporting rapid proliferation and glutathione for providing anti-oxidative stress protection. Evidence obtained from cancer patients and cell lines suggest that NRF2 is highly active in a variety of human cancers and is associated with aggressiveness. p53 is a tumor suppressor that also promotes an anti-oxidative stress metabolic program and glutaminolysis. Here we will dis...
The Plant journal : for cell and molecular biology, 2002
Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene bio... more Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-1-4-endoxylanase activity of the EIX protein further demonstrating that ...
Spatiotemporal modulation of Ras signaling from different intracellular compartments requires mec... more Spatiotemporal modulation of Ras signaling from different intracellular compartments requires mechanisms allowing Ras and its signals to navigate across cells. Here, we describe one mechanism by which clusters of palmitoylated H-Ras and N-Ras isoforms but not nonpalmitoylated K-Ras diffuse through the cytoplasm, independently of ATP, on fast, randomly moving, small cytosolic nanoparticles (''raso- somes''). Rasosomes forced to diffuse out of
Molecular and cellular biology, 2004
The microlocalization of Ras proteins to different microdomains of the plasma membrane is critica... more The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segreg...
The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, a... more The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides.
Methods in Enzymology, 2008
Chronic activation of Ras proteins by mutational activation or by growth factor stimulation is a ... more Chronic activation of Ras proteins by mutational activation or by growth factor stimulation is a common occurrence in many human cancers and was shown to induce and be required for tumor growth. Even if additional genetic defects are present, "correction" of the Ras defect has been shown to reverse Ras-dependent tumorigenesis. One way to block Ras protein activity is by interfering with their spatiotemporal localization in cellular membranes or in membrane microdomains, a prerequisite for Ras signaling and biological activity. Detailed reports describe the use of this method in studies employing farnesylthiosalicylic acid (FTS, Salirasib), a Ras farnesylcysteine mimetic, which selectively disrupts the association of chronically active Ras proteins with the plasma membrane. FTS competes with Ras for binding to Ras-escort proteins, which possess putative farnesyl-binding domains and interact only with the activated form of Ras proteins, thereby promoting Ras nanoclusterization in the plasma membrane and robust signals. This chapter presents three-dimensional time-lapse images that track the FTS-induced inhibition of membrane-activated Ras in live cells on a real-time scale. It also describes a mechanistic model that explains FTS selectivity toward activated Ras. Selective blocking of activated Ras proteins results in the inhibition of Ras transformation in vitro and in animal models, with no accompanying toxicity. Phase I clinical trials have demonstrated a safe profile for oral FTS, with minimal side effects and promising activity in hematological malignancies. Salirasib is currently undergoing trials in patients with pancreatic cancer and with nonsmall cell lung cancer, with or without identified K-Ras mutations. The findings might indicate whether with the disruption of the spatiotemporal localization of oncogenic Ras proteins and the targeting of prenyl-binding domains by anticancer drugs is worth developing as a means of cancer treatment.
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2014
Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment,... more Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment, including hypoxia, nutrient deprivation, and oxidative or genotoxic stress. Tumor cells must evolve adaptive mechanisms to survive these conditions to ultimately drive tumor progression. Tight control of mRNA translation is critical for this response and the adaptation of tumor cells to such stress forms. This proceeds though a translational reprogramming process which restrains overall translation activity to preserve energy and nutrients, but which also stimulates the selective synthesis of major stress adaptor proteins. Here we present the different regulatory signaling pathways which coordinate mRNA translation in the response to different stress forms, including those regulating eIF2α, mTORC1 and eEF2K, and we explain how tumor cells hijack these pathways for survival under stress. Finally, mechanisms for selective mRNA translation under stress, including the utilization of upstream open reading frames (uORFs) and internal ribosome entry sites (IRESes) are discussed in the context of cell stress. This article is part of a Special Issue entitled: Translation and Cancer.
Oncogene, 2014
Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative d... more Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration.
Traffic, 2006
Ras signaling to its downstream effectors appears to include combinations of extracellular-signal... more Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with 'rasosomes', randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function.