Hicham Mahboubi

Nucleolar targeting of the chaperone HSC70 is regulated by stress, cell signaling and a composite targeting signal which is controlled by autoinhibition

Hsc70s are constitutively synthesized members of the 70-kDa chaperone family; they are essential for viability and conserved among all organisms. When eukaryotic cells recover from stress, hsc70s accumulate in nucleoli by an unknown mechanism. Our studies were undertaken to characterize the signaling events and the targeting sequence required to concentrate hsc70 in the nucleoli of human cells. Here, we show that pharmacological inhibitors of phosphatidylinositol (PI) 3-kinase and MEK kinases as well as protein-tyrosine phosphatases abolished the stress-dependent nucleolar accumulation of hsc70. Furthermore, to identify the hsc70 nucleolar targeting sequence, green fluorescent protein-tagged fusion proteins with defined segments of hsc70 were generated and their subcellular distribution was analyzed in growing cells. These studies demonstrated that residues 225 to 297 serve as a heat-inducible nucleolar targeting signal. This segment directs green fluorescent protein to nucleoli in response to stress, but fails to do so under nonstress conditions. Fine mapping of the nucleolar targeting signal revealed that it has two separable functions. First, residues 225 to 262 direct reporter proteins constitutively to nucleoli, even without stress. Second, segment 263 to 287 functions as an autoinhibitory element that prevents hsc70 from concentrating in nucleoli when cells are not stressed. Taken together, PI 3-kinase and MEK kinase signaling as well as tyrosine dephosphorylation are essential for the accumulation of hsc70 in nucleoli of stressed cells. This process relies on a stress-dependent composite targeting signal that combines multiple functions.

Cytoplasmic stress granules: Dynamic modulators of cell signaling and disease

Stress granule (SG) assembly is a conserved cellular strategy to minimize stress-related damage and promote cell survival. Beyond their fundamental role in the stress response, SGs have emerged as key players for human health. As such, SG assembly is associated with cancer, neurodegenerative disorders, ischemia, and virus infections. SGs and granule-related signaling circuits are therefore promising targets to improve therapeutic intervention for several diseases. This is clinically relevant, because pharmacological drugs can affect treatment outcome by modulating SG formation. As membraneless and highly dynamic compartments, SGs regulate translation, ribostasis and proteostasis. Moreover, they serve as signaling hubs that determine cell viability and stress recovery. Various compounds can modulate SG formation and dynamics. Rewiring cell signaling through SG manipulation thus represents a new strategy to control cell fate under various physiological and pathological conditions.

Identification of Novel Stress Granule Components That Are Involved in Nuclear Transport.

Background Importin-α1 belongs to a subfamily of nuclear transport adaptors and participates in diverse cellular functions. Best understood for its role in protein transport, importin-α1 also contributes to other biological processes. For instance, arsenite treatment causes importin-α1 to associate with cytoplasmic stress granules (SGs) in mammalian cells. These stress-induced 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-α subfamilies locate to SGs in response to stress. Results Here, we demonstrate that the oxidant diethyl maleate (DEM), arsenite and heat shock, promote the formation of cytoplasmic SGs that contain nuclear transport factors. Specifically, importin-α1, α4 and α5, which belong to distinct subfamilies, and importin-β1 were targeted by all of these stressors to cytoplasmic SGs, but not to P-bodies. Importin-α family members have been implicated in transcriptional regulation, which prompted us to analyze their ability to interact with poly(A)-RNA in growing cells. Our studies show that importin-α1, but not α4, α5, importin-β1 or CAS, associated with poly(A)-RNA under nonstress conditions. Notably, this interaction was significantly reduced when cells were treated with DEM. Additional studies suggest that importin-α1 is likely connected to poly(A)-RNA through an indirect interaction, as the adaptor did not bind homopolymer RNA specifically in vitro. Significance Our studies establish that members of three importin-α subfamilies are bona fide SG components under different stress conditions. Furthermore, importin-α1 is unique in its ability to interact with poly(A)-RNA in a stress-dependent fashion, and in vitro experiments indicate that this association is indirect. Collectively, our data emphasize that nuclear transport factors participate in a growing number of cellular activities that are modulated by stress.

5'-AMP-activated protein kinase alpha regulates stress granule biogenesis.

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.

Automated detection and quantification of granular cell compartments.

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.

Nucleoli and Stress Granules: Connecting Distant Relatives

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.

Dissecting the molecular mechanisms that impair stress granule formation in aging cells

Aging affects numerous aspects of cell biology, but the senescence-associated changes in the stress response are only beginning to emerge. To obtain mechanistic insights into these events, we examined the formation of canonical and non-canonical stress granules (SGs) in the cytoplasm. SG generation is a key event after exposure to physiological or environmental stressors. It requires the SG-nucleating proteins G3BP1 and TIA-1/TIAR and stress-related signaling events. To analyze SG formation, we used two independent models of somatic cell aging. In both model systems, cellular senescence impaired the assembly of two SG classes: (i) it compromised the formation of canonical SGs, and (ii) skewed the production of non-canonical SGs. We dissected the mechanisms underlying these senescence-dependent changes in granule biogenesis and identified several specific targets that were modulated by aging. Thus, we demonstrate a depletion of G3BP1 and TIA-1/TIAR in senescent cells and show that the loss of G3BP1 contributed to impaired SG formation. We further reveal that aging reduced Sp1 levels; this transcription factor regulated G3BP1 and TIA-1/TIAR abundance. The assembly of canonical SGs relies on the phosphorylation of translation initiation factor eIF2α. We show that senescence can cause eIF2α hyperphosphorylation. CReP is a subunit of protein phosphatase 1 and critical to reverse the stress-dependent phosphorylation of eIF2α. We demonstrate that the loss of CReP correlated with the aging-related hyperphosphorylation of eIF2α. Together, we have identified significant changes in the stress response of aging cells and provide mechanistic insights. Based on our work, we propose that the decline in SG formation can provide a new biomarker to evaluate cellular aging.

Gold Nanoparticles Impinge on Nucleoli and the Stress Response in MCF7 Breast Cancer Cells

Cancer cells can take up gold nanoparticles of different morphologies. These particles interact with the plasma membrane and often travel to intracellular organelles. Among organelles, the nucleus is especially susceptible to the damage that is inflicted by gold nanoparticles. Located inside the nucleus, nucleoli are specialized compartments that transcribe ribosomal RNA genes, produce ribosomes and function as cellular stress sensors. Nucleoli are particularly prone to gold nanoparticle-induced injury. As such, small spherical gold nanoparticles and gold nanoflowers interfere with the transcription of ribosomal DNA. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of gold nanoparticles on nucleolar proteins that are critical to ribosome biogenesis and other cellular functions. We show that B23/nucleophosmin, a nucleolar protein that is tightly linked to cancer, is significantly affected by gold nanoparticles. Furthermore, gold nanoparticles impinge on the cellular stress response, as they reduce the abundance of the molecular chaperone hsp70 and O-GlcNAc modified proteins in the nucleus and nucleoli. Together, our studies set the stage for the development of nanomedicines that target the nucleolus to eradicate proliferating cancer cells.

AMP-kinase Activation Alters Oxidant-induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization

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 physiologic 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, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by 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.

Consistent sex-dependent effects of PKMζ gene ablation and pharmacological inhibition on the maintenance of referred pain.

Background Persistently active PKMζ has been implicated in maintaining spinal nociceptive sensitization that underlies pain hypersensitivity. However, evidence for PKMζ in the maintenance of pain hypersensitivity comes exclusively from short-term studies in males using pharmacological agents of questionable selectivity. The present study examines the contribution of PKMζ to long-lasting allodynia associated with neuropathic, inflammatory, or referred visceral and muscle pain in males and females using pharmacological inhibition or genetic ablation. Results Pharmacological inhibition or genetic ablation of PKMζ reduced mild formalin pain and slowly developing contralateral allodynia in nerve-injured rats, but not moderate formalin pain or ipsilateral allodynia in models of neuropathic and inflammatory pain. Pharmacological inhibition or genetic ablation of PKMζ also effectively reduced referred visceral and muscle pain in male, but not in female mice and rats. Conclusion We show pharmacological inhibition and genetic ablation of PKMζ consistently attenuate long-lasting pain hypersensitivity. However, differential effects in models of referred versus inflammatory and neuropathic pain, and in males versus females, highlight the roles of afferent input-dependent masking and sex differences in the maintenance of pain hypersensitivity.