Analysis of Protein Sumoylation.

Sumoylation, wherein small ubiquitin-like modifier (SUMO) proteins are covalently attached to specific lysine residues of target proteins, plays an important role in regulating many diverse cellular processes via its control of the functional properties of the modified proteins. Identification of new sumoylated proteins is expected to expand understanding of the role this modification has in cell function. This unit describes two different assays for determining whether a particular protein is sumoylated: the first method employs immunoprecipitation of the protein followed by SUMO immunoblot.

Heat shock transcription factor 1 is activated as a consequence of lymphocyte activation and regulates a major proteostasis network in T cells critical for cell division during stress.

Heat shock transcription factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is evoked in response to a variety of cellular stressors, including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.

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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 Huntington's, Alzheimer's, and Parkinson's 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.

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase.

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood.

Detection of proteins sumoylated in vivo and in vitro.

Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating multiple protein functional properties including protein-protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function.

Chromosome-wide analysis of protein binding and modifications.

In order to fully understand the functions of a DNA-binding protein it is necessary to identify all of its binding sites in chromosomes and assess the role of each site in the overall biological function of the factor. An approach ChIP-on-Chip which combines the chromatin immunoprecipitation technique with chromosomal DNA microarray analysis, has proven to be a powerful means for the chromosome-wide identification of protein binding sites. This approach can also be used to characterize chromosome-wide variations in patterns of post-translational protein modifications, for example histone modifications.

RNA polymerase II interacts with the Hspa1b promoter in mouse epididymal spermatozoa.

The Hspa1b (Hsp70.1) gene is one of the first genes expressed after fertilization, with expression occurring during the minor zygotic genome activation (ZGA) in the absence of stress. This expression can take place in the male pronucleus as early as the one-cell stage of embryogenesis.

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, Huntington's, Alzheimer's, and Parkinson's 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.

Identification of a polymorphism in the RING finger of human Bmi-1 that causes its degradation by the ubiquitin-proteasome system.

Bmi-1 is a polycomb protein that plays an important role in tumor cell development and maintaining stem cell populations of many cell lineages. Here we identify a polymorphism in human Bmi-1 that changes a cysteine within its RING domain to tyrosine. This C18Y polymorphism is associated with a significant decrease in Bmi-1 level and its elevated ubiquitination, suggesting that it is being destroyed by the ubiquitin-proteasome system.

Phosphorylation of CAP-G is required for its chromosomal DNA localization during mitosis.

Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation.

Detection of sumoylated proteins.

Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating a variety of different protein functional properties, including protein-protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as of nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function.

The TBP-PP2A mitotic complex bookmarks genes by preventing condensin action.

To maintain phenotypes of cell lineages, cells must 'remember' which genes were active before mitosis entry and transmit this information to their daughter cells so that expression patterns can be faithfully re-established in G1. This phenomenon is called gene bookmarking. However, during mitosis transcription ceases, most sequence-specific proteins dissociate from DNA and the chromatin is tightly compacted, making it difficult to understand how gene activity 'memory' is maintained through this stage of the cell cycle.

Mel-18 interacts with RanGAP1 and inhibits its sumoylation.

Our previous results showed that the polycomb protein mel-18 binds to a protein called HSF2 and inhibits HSF2 sumoylation, thereby functioning as an anti-SUMO E3 factor. This study also suggested that mel-18 regulates the sumoylation of other cellular proteins, but the identities of these other proteins were unknown. Here we show that mel-18 interacts with the RanGAP1 protein and inhibits its sumoylation, and that these activities do not require the RING domain of mel-18.

Sumoylation of amyloid precursor protein negatively regulates Abeta 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 Alzheimer's 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.

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.

PRC1 associates with the hsp70i promoter and interacts with HSF2 during mitosis.

Mitosis is a series of events leading to division of a cell by the process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a CDK substrate that associates with the mitotic spindle and functions in microtubule bundling. Previous studies revealed that loss of PRC1 is associated with chromosomal mis-segregation and atypical chromosome alignment.

Interaction of HSF1 and HSF2 with the Hspa1b promoter in mouse epididymal spermatozoa.

The Hspa1b gene is one of the first genes expressed after fertilization, with expression observed in the male pronucleus as early as the one-cell stage of embryogenesis. This expression can occur in the absence of stress and is initiated during the minor zygotic genome activation. There is a significant reduction in the number of embryos developing to the blastocyte stage when HSPA1B levels are depleted, which supports the importance of this protein for embryonic viability.

Analysis of protein sumoylation.

The covalent attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues of target proteins, a process termed sumoylation, is a recently discovered protein modification that plays an important role in regulating many diverse cellular processes. For this reason there is significant interest in identifying new sumoylated proteins and the lysine residue(s) within these target proteins where SUMO attachment occurs. Such knowledge will allow determination of the functional consequences of sumoylation through mutation of the relevant sequences.

MEL-18 interacts with HSF2 and the SUMO E2 UBC9 to inhibit HSF2 sumoylation.

In a previous study we found that sumoylation of the DNA-binding protein heat shock factor 2 (HSF2) is up-regulated during mitosis, but the mechanism that mediates this regulation was unknown. Here we show that HSF2 interacts with the polycomb protein MEL-18, that this interaction decreases during mitosis, and that overexpression and RNA interference-mediated reduction of MEL-18 result in decreased and increased HSF2 sumoylation, respectively. Other results suggest that MEL-18 may also function to inhibit the sumoylation of other cellular proteins.

Physiological fever temperature induces a protective stress response in T lymphocytes mediated by heat shock factor-1 (HSF1).

Heat shock factor-1 (HSF1) is a transcription factor that serves as the major temperature-inducible sensor for eukaryotic cells. In most cell types, HSF1 becomes activated to the DNA binding form at 42 degrees C and mediates the classical heat shock response, protecting the cells from subsequent lethal temperatures. We have recently demonstrated that HSF1 is activated at a lower temperature in T lymphocytes than in most other cell types (39 degrees C vs 42 degrees C), within the physiological range of fever.

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.

HSF2 binds to the Hsp90, Hsp27, and c-Fos promoters constitutively and modulates their expression.

Although the vast majority of genomic DNA is tightly compacted during mitosis, the promoter regions of a number of genes remain in a less compacted state throughout this stage of the cell cycle. The decreased compaction of these promoter regions, which is referred to as gene bookmarking, is thought to be important for the ability of cells to express these genes during the following interphase. Previously, we reported a role for the DNA-binding protein heat shock factor (HSF2) in bookmarking the stress-inducible 70,000-Da heat shock protein (hsp70) gene.

HSF1-TPR interaction facilitates export of stress-induced HSP70 mRNA.

Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear pore-associating TPR protein in a stress-responsive manner.