Hicham Mahboubi | Novartis - Academia.edu (original) (raw)

Uploads

Papers by Hicham Mahboubi

Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Differe... more Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK) which functions as a stress sensor and is transiently activated by adverse physiological conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, as the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, the abundance of translation initiation factors and cytoskeletal organization. Specifically, AMPK activation increased the stress-induced eIF2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of protein deacetylase HDAC6 was diminished. This loss of HDAC6 was accompanied by an increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation, but limits their fusion into larger granules.

Microscopy and Microanalysis, 2013

Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effect... more Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules~SGs!. To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming, while preventing the efficient use of high-throughput technology. We developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Our methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of our protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, we show that the type of stress determines granule size and composition, as illustrated by the concentration of poly~A!-RNA and a specific SG marker protein. Furthermore, we took advantage of the computer-based and automated methods to design assays suitable for high-throughput screening.

Journal of Biological Chemistry, 2011

4 The abbreviations used are: Hsc70, heat shock cognate protein 70; PI, phosphatidylinositol; DMS... more 4 The abbreviations used are: Hsc70, heat shock cognate protein 70; PI, phosphatidylinositol; DMSO, dimethyl sulfoxide; GFP, humanized green fluorescent protein; NPC, nuclear pore complex; NLS, nuclear localization sequence; NoLS, nucleolar localization sequence; PAO, phenylarsine oxide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase.

Traffic (Copenhagen, Denmark), 2014

Nucleoli and cytoplasmic stress granules (SGs) are subcellular compartments that modulate the res... more Nucleoli and cytoplasmic stress granules (SGs) are subcellular compartments that modulate the response to endogenous and environmental signals to control cell survival. In our opinion, nucleoli and SGs are functionally linked; they are distant relatives that combine forces when cellular homeostasis is threatened. Several lines of evidence support this idea; nucleoli and SGs share molecular building blocks, are regulated by common signaling pathways and communicate when vital cellular functions become compromised. Together, nucleoli and SGs orchestrate physiological responses that are directly relevant to stress and human health. As both compartments have established roles in neurodegenerative diseases, cancer and virus infections, we propose that these conditions will benefit from therapeutic interventions that target simultaneously nucleoli and SGs.

Farooqui/Metabolic, 2013

ABSTRACT 5’-AMP activated kinase (AMPK) serves as an energy sensor that is implicated in numerous... more ABSTRACT 5’-AMP activated kinase (AMPK) serves as an energy sensor that is implicated in numerous biological processes. This ser/thr protein kinase provides a focal point for metabolic control in all eukaryotes, where it exerts essential functions in different organs and cell types. Due to its critical function in metabolism, AMPK has been recognized as a key player in many human diseases and disorders. Thus, as a crucial regulator of glucose, lipid, and protein homeostasis, AMPK is now an important therapeutic target for type 2 diabetes, obesity, and cancer. Moreover, the kinase is also emerging as a promising target for therapeutic intervention in multiple neurodegenerative diseases. This chapter discusses the specific roles that AMPK plays in different organs and tissues, with emphasis on those that are particularly relevant to human health. To this end, we will focus on the processes that link AMPK to type 2 diabetes and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease.

PLoS ONE, 2013

Background: Importin-a1 belongs to a subfamily of nuclear transport adaptors and participates in ... more Background: Importin-a1 belongs to a subfamily of nuclear transport adaptors and participates in diverse cellular functions. Best understood for its role in protein transport, importin-a1 also contributes to other biological processes. For instance, arsenite treatment causes importin-a1 to associate with cytoplasmic stress granules (SGs) in mammalian cells. These stressinduced compartments contain translationally arrested mRNAs, small ribosomal subunits and numerous proteins involved in mRNA transport and metabolism. At present, it is not known whether members of all three importin-a subfamilies locate to SGs in response to stress.

Background. Chaperones and their co-factors are components of a cellular network; they collabora... more Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities.

Methods and Results. Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i) the physiological state of the cell and (ii) the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm.

Discussion. Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1.

Data in Brief (Elsevier), May 5, 2015

This data article contains insights into the regulation of cytoplasmic stress granules (SGs) by 5... more This data article contains insights into the regulation of cytoplasmic stress granules (SGs) by 5′-AMP-activated kinase (AMPK). Our results verify the specific association of AMPK-α2, but not AMPK-α1, with SGs. We also provide validation data for the isoform-specific recruitment of the AMPK-α subunit to SGs using (i) different antibodies and (ii) a distinct cellular model system. In addition, we assess the SG association of the regulatory AMPK β- and γ-subunits. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in “5’AMP-activated protein kinase alpha regulates stress granule biogenesis”.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Mar 31, 2015

AMPK-α2 is a bona fide component of cytoplasmic stress granules.AMPK-α controls the biogenesis of... more AMPK-α2 is a bona fide component of cytoplasmic stress granules.AMPK-α controls the biogenesis of cytoplasmic stress granules.AMPK-α2 binds directly to the granule nucleation protein G3BP1 in stress granules.Stress stimulates the interaction between AMPK-α2 and G3BP1.Stress granule (SG) assembly represents a conserved eukaryotic defense strategy against various insults. Although essential for the ability to cope with deleterious conditions, the signaling pathways controlling SG formation are not fully understood. The energy sensor AMP-activated protein kinase (AMPK) is critical for the cellular stress response. Human cells produce two AMPK catalytic α-subunits with not only partially overlapping, but also unique functions. Here, we provide direct support for structural and functional links between AMPK-α isoforms and SGs. As such, several stressors promote SG association of AMPK-α2, but not AMPK-α1. Multiple lines of evidence link AMPK activity to SG biogenesis. First, pharmacological kinase inhibition interfered with SG formation. Second, AMPK-α knockdown combined with in-depth quantitative SG analysis revealed isoform-specific changes of SG characteristics. Third, overexpression of mutant α-subunits further substantiated that AMPK regulates SG parameters. Finally, we identified the SG-nucleating protein G3BP1 as an AMPK-α2 binding partner. This interaction is stimulated by stress and notably occurs in SGs. Collectively, our data define the master metabolic regulator AMPK as a novel SG constituent that also controls their biogenesis.

Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Differe... more Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK) which functions as a stress sensor and is transiently activated by adverse physiological conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, as the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, the abundance of translation initiation factors and cytoskeletal organization. Specifically, AMPK activation increased the stress-induced eIF2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of protein deacetylase HDAC6 was diminished. This loss of HDAC6 was accompanied by an increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation, but limits their fusion into larger granules.

Microscopy and Microanalysis, 2013

Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effect... more Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules~SGs!. To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming, while preventing the efficient use of high-throughput technology. We developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Our methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of our protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, we show that the type of stress determines granule size and composition, as illustrated by the concentration of poly~A!-RNA and a specific SG marker protein. Furthermore, we took advantage of the computer-based and automated methods to design assays suitable for high-throughput screening.

Journal of Biological Chemistry, 2011

4 The abbreviations used are: Hsc70, heat shock cognate protein 70; PI, phosphatidylinositol; DMS... more 4 The abbreviations used are: Hsc70, heat shock cognate protein 70; PI, phosphatidylinositol; DMSO, dimethyl sulfoxide; GFP, humanized green fluorescent protein; NPC, nuclear pore complex; NLS, nuclear localization sequence; NoLS, nucleolar localization sequence; PAO, phenylarsine oxide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase.

Traffic (Copenhagen, Denmark), 2014

Nucleoli and cytoplasmic stress granules (SGs) are subcellular compartments that modulate the res... more Nucleoli and cytoplasmic stress granules (SGs) are subcellular compartments that modulate the response to endogenous and environmental signals to control cell survival. In our opinion, nucleoli and SGs are functionally linked; they are distant relatives that combine forces when cellular homeostasis is threatened. Several lines of evidence support this idea; nucleoli and SGs share molecular building blocks, are regulated by common signaling pathways and communicate when vital cellular functions become compromised. Together, nucleoli and SGs orchestrate physiological responses that are directly relevant to stress and human health. As both compartments have established roles in neurodegenerative diseases, cancer and virus infections, we propose that these conditions will benefit from therapeutic interventions that target simultaneously nucleoli and SGs.

Farooqui/Metabolic, 2013

ABSTRACT 5’-AMP activated kinase (AMPK) serves as an energy sensor that is implicated in numerous... more ABSTRACT 5’-AMP activated kinase (AMPK) serves as an energy sensor that is implicated in numerous biological processes. This ser/thr protein kinase provides a focal point for metabolic control in all eukaryotes, where it exerts essential functions in different organs and cell types. Due to its critical function in metabolism, AMPK has been recognized as a key player in many human diseases and disorders. Thus, as a crucial regulator of glucose, lipid, and protein homeostasis, AMPK is now an important therapeutic target for type 2 diabetes, obesity, and cancer. Moreover, the kinase is also emerging as a promising target for therapeutic intervention in multiple neurodegenerative diseases. This chapter discusses the specific roles that AMPK plays in different organs and tissues, with emphasis on those that are particularly relevant to human health. To this end, we will focus on the processes that link AMPK to type 2 diabetes and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease.

PLoS ONE, 2013

Background: Importin-a1 belongs to a subfamily of nuclear transport adaptors and participates in ... more Background: Importin-a1 belongs to a subfamily of nuclear transport adaptors and participates in diverse cellular functions. Best understood for its role in protein transport, importin-a1 also contributes to other biological processes. For instance, arsenite treatment causes importin-a1 to associate with cytoplasmic stress granules (SGs) in mammalian cells. These stressinduced compartments contain translationally arrested mRNAs, small ribosomal subunits and numerous proteins involved in mRNA transport and metabolism. At present, it is not known whether members of all three importin-a subfamilies locate to SGs in response to stress.

Background. Chaperones and their co-factors are components of a cellular network; they collabora... more Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities.

Methods and Results. Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i) the physiological state of the cell and (ii) the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm.

Discussion. Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1.

Data in Brief (Elsevier), May 5, 2015

This data article contains insights into the regulation of cytoplasmic stress granules (SGs) by 5... more This data article contains insights into the regulation of cytoplasmic stress granules (SGs) by 5′-AMP-activated kinase (AMPK). Our results verify the specific association of AMPK-α2, but not AMPK-α1, with SGs. We also provide validation data for the isoform-specific recruitment of the AMPK-α subunit to SGs using (i) different antibodies and (ii) a distinct cellular model system. In addition, we assess the SG association of the regulatory AMPK β- and γ-subunits. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in “5’AMP-activated protein kinase alpha regulates stress granule biogenesis”.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Mar 31, 2015

AMPK-α2 is a bona fide component of cytoplasmic stress granules.AMPK-α controls the biogenesis of... more AMPK-α2 is a bona fide component of cytoplasmic stress granules.AMPK-α controls the biogenesis of cytoplasmic stress granules.AMPK-α2 binds directly to the granule nucleation protein G3BP1 in stress granules.Stress stimulates the interaction between AMPK-α2 and G3BP1.Stress granule (SG) assembly represents a conserved eukaryotic defense strategy against various insults. Although essential for the ability to cope with deleterious conditions, the signaling pathways controlling SG formation are not fully understood. The energy sensor AMP-activated protein kinase (AMPK) is critical for the cellular stress response. Human cells produce two AMPK catalytic α-subunits with not only partially overlapping, but also unique functions. Here, we provide direct support for structural and functional links between AMPK-α isoforms and SGs. As such, several stressors promote SG association of AMPK-α2, but not AMPK-α1. Multiple lines of evidence link AMPK activity to SG biogenesis. First, pharmacological kinase inhibition interfered with SG formation. Second, AMPK-α knockdown combined with in-depth quantitative SG analysis revealed isoform-specific changes of SG characteristics. Third, overexpression of mutant α-subunits further substantiated that AMPK regulates SG parameters. Finally, we identified the SG-nucleating protein G3BP1 as an AMPK-α2 binding partner. This interaction is stimulated by stress and notably occurs in SGs. Collectively, our data define the master metabolic regulator AMPK as a novel SG constituent that also controls their biogenesis.