SUMO-specific proteases: a twist in the tail

The small ubiquitin-like modifier (SUMO) is involved in many cellular processes and is required for normal growth and development in all eukaryotes. Whereas lower eukaryotes have a single version of SUMO, higher eukaryotes have three versions: SUMO-1, -2 and -3. Similarly to most other ubiquitin-like proteins, the primary translation products of the SUMO genes need to be proteolytically processed to expose the C-terminal glycine that will be linked to lysine side chains in substrates. Processing of SUMO precursors is mediated by SUMO-specific proteases that also remove SUMO from modified proteins and depolymerise poly-SUMO chains.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

The dynamics and mechanism of SUMO chain deconjugation by SUMO-specific proteases

SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system. We show that SUMO-specific proteases (SENPs) play a crucial role in the dynamics of SUMO chains in vivo by constant deconjugation. Preventing deSUMOylation in Schizosaccharomyces pombe results in slow growth and a sensitivity to replication stress, highlighting the biological requirement for deSUMOylation dynamics. Furthermore, we present the mechanism of SUMO chain deconjugation by SENPs, which occurs via a stochastic mechanism, resulting in cleavage anywhere within a chain. Our results offer mechanistic insights into the workings of deSUMOylating proteases and highlight their importance in the homeostasis of (poly)SUMO-modified substrates.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Characterization of a family of nucleolar SUMO-specific proteases with preference for SUMO-2 or SUMO-3

SUMOylation is a reversible process regulated by a family of sentrin/SUMO-specific proteases (SENPs). Of the six SENP family members, except for SENP1 and SENP2, the substrate specificities of the rest of SENPs are not well defined. Here, we have described SENP5, which has restricted substrate specificity. SENP5 showed SUMO-3 C-terminal hydrolase activity but could not process pro-SUMO-1 in vitro. Furthermore, SENP5 showed more limited isopeptidase activity in vitro. In vivo, SENP5 showed isopeptidase activity against SUMO-2 and SUMO-3 conjugates but not against SUMO-1 conjugates. Native SENP5 localized mainly to the nucleolus but was also found in the nucleus. The N terminus of SENP5 contains a stretch of amino acids responsible for the nucleolar localization of SENP5. N-terminal-truncated SENP5 co-localized with PML, a known SUMO substrate. Using PML SUMOylation mutants as model substrates, we showed that SENP5 can remove poly-SUMO-2 or poly-SUMO-3 from the Lys160 or Lys490 positions of PML. However, SENP5 could not remove SUMO-1 from the Lys160 or Lys490 positions of PML. Nonetheless, SENP5 could remove SUMO-1, -2, and -3 from the Lys65 position of PML. Thus, SENP5 also possesses limited SUMO-1 isopeptidase activity. We were also able to show that SENP3 has substrate specificity similar to that of SENP5. Thus, SENP3 and SENP5 constitute a subfamily of SENPs that regulate the formation of SUMO-2 or SUMO-3 conjugates and, to a less extent, SUMO-1 modification.     

Proteasome inhibition induces differential heat shock protein response but not unfolded protein response in HepG2 cells

Liver, a central organ responsible for the metabolism of carbohydrates, proteins, and lipoproteins, is exposed to various kinds of physiological, pathological, and environmental stresses. We hypothesized that blockage of proteasome degradation pathway induces heat shock protein (HSP) response and unfolded protein response in the liver cells. In this study, we have characterized cellular responses to proteasome inhibition in HepG2 cells, a well-differentiated human hepatoma cells. We found that proteasome inhibition induced differential response among cytosolic HSPs, that is, increased expression of HSP70, but no change in HSP40, HSC70, and HSP90. However, proteasome inhibition did not induce typical unfolded protein response as indicated by absence of stimulation of GRP78 and GRP94 proteins. Upon proteasome inhibition, inclusion bodies were accumulated, and ubiquitin-conjugated proteins appeared in insoluble fraction, together with HSP40, HSP70, HSC70, and HSP90. After proteasome inhibition, misfolded proteins were increased in the cytosol and in the ER compartment as evaluated by examining ubiquitin-conjugated proteins. However, essentially all ER-associated ubiquitin-conjugated proteins were located on the surface of the ER, which explains why proteasome inhibition does not induce unfolded protein response. In conclusion, proteasome inhibition induces differential HSP response, but not unfolded protein response in HepG2 cells. Our study also suggests that HSPs play important roles in directing proteasomal degradation and protein aggregate formation.     

Heat shock induces variably the major heat shock proteins of CV1 clones

CV1 cells have been subcloned several times. Five of these clones were studied for the induction of the major heat shock proteins. These CV1 clones exhibit morphological differences as well as differences in SDS-PAGE protein profiles. These clones responded to heat shock variably as judged from the induction of the major heat shock proteins, 70, 72 and 92 kDa. Variable expression of the heat shock proteins suggests that the selective pressure for isolation of cell clones may affect gene expression differently.     

Heat shock induces protein tyrosine phosphorylation in cultured cells

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.     

Heat shock induces chromosome loss in the yeast Candida albicans

Summary The heat shock protocol described in this paper causes mitotic instability in log phase Candida albicans cells. Such instability is induced in diploid, aneuploid and tetraploid strains. The strains analysed are multiple heterozygotes which facilitates the detection of mitotic instability as manifested by the formation of homozygotes. Strains previously shows to be carrying cis linked mutant alleles show coincident segregation of the linked alleles. Conversely, strains which carry unlinked mutant alleles display no such coincident segregation. This segregation of complete linkage groups suggests that heat shock is inducing chromosome some loss in C. albicans. The application of this protocol to the genetics of the imperfect fungus C. albicans has produced evidence of at least three chromosomes.     

Heat shock protein 60 induces inflammatory mediators in mouse adipocytes

Adipocytes represent an important cellular source of inflammatory mediators. However, the signals for the induction of proinflammatory adipocyte activities are largely unknown. Here, we demonstrate that heat shock protein (Hsp) 60, a potent stimulator of innate immunity, induces the release of the inflammatory mediators interleukin-6, CXCL1 and monocyte chemoattractant protein-1 in a time- and concentration-dependent manner from cells of the adipocyte line 3T3-L1 and from adipocytes of obese mice. These results identify Hsp60 as an important regulator of adipocyte functions which contribute to the development of inflammatory processes as observed in diabetes and diabetes-associated complications.     

Heat shock induces apoptosis independently of any known initiator caspase-activating complex

Adaptive responses to mild heat shock are among the most widely conserved and studied in nature. More intense heat shock, however, induces apoptosis through mechanisms that remain largely unknown. Herein, we present evidence that heat shock activates an apical protease that stimulates mitochondrial outer membrane permeabilization and processing of the effector caspase-3 in a benzyloxycarbonyl-VAD-fluoromethyl ketone (polycaspase inhibitor)- and Bcl-2-inhibitable manner. Surprisingly, however, neither FADD.caspase-8 nor RAIDD.caspase-2 PIDDosome (p53-induced protein with a death domain) complexes were detected in dying cells, and neither of these initiator caspases nor the endoplasmic reticulum stress-activated caspases-4/12 were required for mitochondrial outer membrane permeabilization. Similarly, although cytochrome c was released from mitochondria following heat shock, functional Apaf-1.caspase-9 apoptosome complexes were not formed, and caspase-9 was not essential for the activation of caspase-3 or the induction of apoptosis. Thus, heat shock does not require any of the known initiator caspases or their activating complexes to promote apoptotic cell death but instead relies upon the activation of an apparently novel apical protease with caspase-like activity.     

Heat shock induces intestinal-type alkaline phosphatase in rat IEC-18 cells

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.     

Heat shock induces premature transcript termination and reconfigures the human transcriptome

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Heat shock induces alterations of the lipoxygenase pathway in human polymorphonuclear granulocytes

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.     

Heat shock induces phosphorylation of histone H2AX in mammalian cells

Heat shock induces a variety of biological events including gene activation, cell cycle arrest, and apoptosis. Heat shock has recently been shown to be potentially useful when combined with radiation in cancer therapy, probably because, in mammalian cells, heat inhibits the repair of double-strand breaks (DSBs) induced by ionizing radiation. It remains unclear, however, whether heat shock by itself induces DSBs. In this communication, we present the first evidence that heat shock induces the phosphorylated form of histone H2AX, which is thought to be generated at the chromatin proximal to DSB sites. These results suggest that heat shock induces DSBs in mammalian cells and may provide direct evidence to explain previous reports on DSB-related events occurring after heat shock treatment.     

SIRT6 overexpression induces massive apoptosis in cancer cells but not in normal cells

Emerging evidence suggests that Sirtuin 6 (SIRT6) functions as a longevity assurance gene by promoting genomic stability, regulating metabolic processes and attenuating inflammation. Here, we examine the effect of SIRT6 activation on cancer cells. We show that SIRT6 overexpression induces massive apoptosis in a variety of cancer cell lines but not in normal, non-transformed cells. This cell death requires the mono-ADP-ribosyltransferase but not the deacetylase activity of SIRT6 and is mediated by the activation of both the p53 and p73 apoptotic signaling cascades in cancer cells by SIRT6. These results suggest that SIRT6 is an attractive target for pharmacological activation in cancer treatment.     

SIRT6 overexpression induces massive apoptosis in cancer cells but not in normal cells

Emerging evidence suggests that Sirtuin 6 (SIRT6) functions as a longevity assurance gene by promoting genomic stability, regulating metabolic processes and attenuating inflammation. Here, we examine the effect of SIRT6 activation on cancer cells. We show that SIRT6 overexpression induces massive apoptosis in a variety of cancer cell lines but not in normal, non-transformed cells. This cell death requires the mono-ADP-ribosyltransferase but not the deacetylase activity of SIRT6 and is mediated by the activation of both the p53 and p73 apoptotic signaling cascades in cancer cells by SIRT6. These results suggest that SIRT6 is an attractive target for pharmacological activation in cancer treatment.Key words: cancer, SIRT6, p53, p73, DNA damage