Kevin D. Sarge

SUMO-1 Modification Regulates the DNA Binding Activity of Heat Shock Transcription Factor 2, a Promyelocytic Leukemia Nuclear Body Associated Transcription Factor

Heat shock transcription factor 2 (HSF2) is a transcription factor that regulates heat shock protein gene expression, but the mechanisms regulating the function of this factor are unclear. Here we report that HSF2 is a substrate for modification by the ubiquitin-related protein SUMO-1 and that HSF2 colocalizes in cells with SUMO-1 in nuclear granules. Staining with anti-promyelocytic leukemia antibodies indicates that these HSF2-containing nuclear granules are PML bodies. Our results identify lysine 82 as the major site of SUMO-1 modification in HSF2, which is located in a “wing” within the DNA-binding domain of this protein. Interestingly, SUMO-1 modification of HSF2 results in conversion of this factor to the active DNA binding form. This is the first demonstration that SUMO-1 modification can directly alter the DNA binding ability of a transcription factor and reveals a new mechanism by which SUMO-1 modification can regulate protein function.

Sumoylation regulates lamin A function and is lost in lamin A mutants associated with familial cardiomyopathies.

Lamin A mutations cause many diseases, including cardiomyopathies and Progeria Syndrome. The covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides regulates the function of many proteins. Until now, no examples of human disease-causing mutations that occur within a sumoylation consensus sequence and alter sumoylation were known. We show that lamin A is sumoylated at lysine 201 and that two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. E203 occupies the conserved +2 position in the sumoylation consensus ΨKXE. Lamin A mutants E203G, E203K, and K201R all exhibit a similar aberrant subcellular localization and are associated with increased cell death. Fibroblasts from an individual with the E203K lamin A mutation also exhibit decreased lamin A sumoylation and increased cell death. These results suggest that SUMO modification is important for normal lamin A function and implicate an involvement for altered sumoylation in the E203G/E203K lamin A cardiomyopathies.

Sumoylation and human disease pathogenesis

Covalent modification by SUMO polypeptides, or sumoylation, is an important regulator of the functional properties of many proteins. Among these are several proteins implicated in human diseases including cancer, Huntingtons, Alzheimers, and Parkinsons diseases, as well as spinocerebellar ataxia 1 and amyotrophic lateral sclerosis. Recent reports reveal two new examples of human disease-associated proteins that are SUMO modified: amyloid precursor protein and lamin A. These findings point to a function for sumoylation in modulating amyloid-beta peptide levels, indicating a potential role in Alzheimers disease, and for decreased lamin A sumoylation as a causative factor in familial dilated cardiomyopathy.

Effect of caloric restriction on the expression of heat shock protein 70 and the activation of heat shock transcription factor 1

The regulation of heat shock protein 70 (hsp70) expression is an excellent example of a cellular mechanism that has evolved to protect all living organisms from various types of physiological stresses; therefore, the reported age-related alterations in the ability of cells to express hsp70 in response to stress could seriously compromise the ability of a senescent organism in respond to changes in its environment. Because caloric restriction (CR) is the only experimental manipulation known to retard aging and increase the survival of rodents, it was of interest to analyze the effect of CR on the age-related alteration in the induction of hsp70 expression in rat hepatocytes. The effect of CR on the nuclear transcription of hsp70 gene in rat hepatocytes in response to various levels of heat shock was determined, and it was found that the age-related decline in the transcription of hsp70 at all temperatures studied was reversed by CR. Because the heat shock transcription factor (HSF) mediates the heat-induced transcription of hsp70, the effect of CR on the induction of HSF binding activity by heat shock was studied and found to arise from HSF1, which has been shown to be involved in the induction of HSF binding activity in other cell types. The age-related decrease in the induction of HSF1 binding activity in rat hepatocytes was reversed by CR, and did not appear to be due to an accumulation of inhibitory molecules with age. Interestingly, the level of HSF1 protein was significantly higher in hepatocytes isolated from old rats fed ad libitum compared to hepatocytes obtained from rats fed the CR diet even though the levels of HSF1 binding activity were lower for hepatocytes isolated from the old rats fed ad libitum. The levels of the mRNA transcript for HSF1 was not significantly altered by age or CR. Thus, the changes in HSF1 binding activity with age and CR do not arise from changes in the level of HSF1 protein available for activation.

Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities.

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.

Celastrol inhibits polyglutamine aggregation and toxicity though induction of the heat shock response

Heat shock proteins (hsps) are protective against the harmful effects of mutant expanded polyglutamine repeat proteins that occur in diseases such as Huntington’s, prompting the search for pharmacologic compounds that increase hsp expression in cells as potential treatments for this and related diseases. In this paper, we show that celastrol, a compound recently shown to up-regulate hsp gene expression, significantly decreases killing of cells expressing mutant polyglutamine protein. This effect requires the presence of the transcription factor responsible for mediating inducible hsp gene expression, HSF1, and is correlated with decreased amounts and increased sodium dodecyl sulfate (SDS) solubility of polyglutamine aggregates. These results suggest the potential of celastrol as a therapeutic agent in the treatment of human polyglutamine expansion diseases.

Characterization of Hypothermia-induced Cellular Stress Response in Mouse Tissues

Cells respond to adverse environmental conditions by expressing heat shock proteins, which serve to protect cells from harmful effects of the stress conditions. In this study we demonstrated that mice subjected to whole body hypothermia induced the cellular stress response, resulting in the increased expression of hsp72 mRNA in brain, heart, kidney, liver, and lung. We performed a detailed analysis of the major parameters of the stress response and found that cold induction of hsp expression is mediated by heat shock factor 1 (HSF1), which is also responsible for heat induction of the cellular stress response. However, there are differences in the mechanisms of HSF1 activation by hypothermia versus hyperthermia, as hypothermia does not cause the hyperphosphorylation of HSF1 that is characteristic of heat-activated HSF1.

Sumoylation of amyloid precursor protein negatively regulates Aβ aggregate levels

The proteolytic processing of amyloid precursor protein (APP) to produce Abeta peptides is thought to play an important role in the mechanism of Alzheimers disease. Here, we show that lysines 587 and 595 of APP, which are immediately adjacent to the site of beta-secretase cleavage, are covalently modified by SUMO proteins in vivo. Sumoylation of these lysine residues is associated with decreased levels of Abeta aggregates. Further, overexpression of the SUMO E2 enzyme ubc9 along with SUMO-1 results in decreased levels of Abeta aggregates in cells transfected with the familial Alzheimers disease-associated V642F mutant APP, indicating the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimers disease. The results also provide the first demonstration that the SUMO E2 enzyme (ubc9) is present within the endoplasmic reticulum, indicating how APP, and perhaps other proteins that enter this compartment, can be sumoylated.

SUMO and Its Role in Human Diseases

The covalent attachment of small ubiquition-like modifier (SUMO) polypeptides, or sumoylation, is an important regulator of the functional properties of many proteins. Among these are many proteins implicated in human diseases including cancer and Huntingtons, Alzheimers, and Parkinsons diseases, as well as spinocerebellar ataxia 1 and amyotrophic lateral sclerosis. The results of two more recent studies identify two additional human disease-associated proteins that are sumoylated, amyloid precursor protein (APP), and lamin A. APP sumoylation modulates Aβ peptide levels, suggesting a potential role in Alzheimers disease, and decreased lamin A sumoylation due to mutations near its SUMO site has been implicated in causing some forms of familial dilated cardiomyopathy.

Interaction between Protein Phosphatase 5 and the A subunit of Protein Phosphatase 2A EVIDENCE FOR A HETEROTRIMERIC FORM OF PROTEIN PHOSPHATASE 5

Members of the phosphoprotein phosphatase family of serine/threonine phosphatases are thought to exist in different native oligomeric complexes. Protein phosphatase 2A (PP2A) is composed of a catalytic subunit (PP2Ac) that complexes with an A subunit, which in turn also interacts with one of many B subunits that regulate substrate specificity and/or (sub)cellular localization of the enzyme. Another family member, protein phosphatase 5 (PP5), contains a tetratricopeptide repeat domain at its N terminus, which has been suggested to mediate interactions with other proteins. PP5 was not thought to interact with partners homologous to the A or B subunits that exist within PP2A. However, our results indicate that this may not be the case. A yeast two-hybrid screen revealed an interaction between PP5 and the A subunit of PP2A. This interaction was confirmed for endogenous proteins in vivo using immunoprecipitation analysis and for recombinant proteins by in vitro binding experiments. Our results also indicate that the tetratricopeptide repeat domain of PP5 is required and sufficient for this interaction. In addition, immunoprecipitated PP5 contains associated B subunits. Thus, our results suggest that PP5 can exist in a PP2A-like heterotrimeric form containing both A and B subunits.