Ubiquitination Occurs in the Mitochondrial Matrix by Eclipsed Targeted Components of the Ubiquitination Machinery.

Ubiquitination is a critical type of post-translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria.

Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation.

The Krebs cycle enzyme fumarase, which has been identified as a tumor suppressor, is involved in the deoxyribonucleic acid (DNA) damage response (DDR) in human, yeast, and bacterial cells. We have found that the overexpression of the cysteine desulfurase Nfs1p restores DNA repair in fumarase-deficient yeast cells. Nfs1p accumulates inactivating post-translational modifications in yeast cells lacking fumarase under conditions of DNA damage.

A combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response in .

Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR.

Post-translational Modifications of Fumarase Regulate its Enzyme Activity and Function in Respiration and the DNA Damage Response.

The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea).

Fumarase: From the TCA Cycle to DNA Damage Response and Tumor Suppression.

Fumarase is an enzyme of the tricarboxylic acid (TCA) cycle in mitochondria, but in recent years, it has emerged as a participant in the response to DNA double strand breaks (DSBs) in the nucleus. In fact, this enzyme is dual-targeted and can be also readily detected in the mitochondrial and cytosolic/nuclear compartments of all the eukaryotic organisms examined. Intriguingly, this evolutionary conserved cytosolic population of fumarase, its enzymatic activity and the associated metabolite fumarate, are required for the cellular DNA damage response (DDR) to double-strand breaks.

Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2.

One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the main DSB repair pathways.

Human Fumarate Hydratase Is Dual Localized by an Alternative Transcription Initiation Mechanism.

Fumarate hydratase (FH, fumarase), is a tricarboxylic acid cycle enzyme localized in the mitochondrial matrix. However, a common theme, conserved from yeast to human, is the existence of a large cytosolic population of FH. FH has been shown to function as a tumor suppressor gene and is now implicated in various diseases.

Differential regulation of proinflammatory cytokine expression by mitogen-activated protein kinases in macrophages in response to intestinal parasite infection.

Blastocystis is a common enteric protistan parasite that can cause acute, as well as chronic, infection and is associated with irritable bowel syndrome (IBS). However, the pathogenic status of Blastocystis infection remains unclear. In this study, we found that Blastocystis antigens induced abundant expression of proinflammatory cytokines, including interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), in mouse intestinal explants, in mouse colitis colon, and in macrophages.

How the ubiquitin proteasome system regulates the regulators of transcription.

The ubiquitin proteasome system plays an important role in transcription. Monoubiquitination of activators is believed to aid their function, while the 26S proteasomal degradation of repressors is believed to restrict their function. What remains controversial is the question of whether the degradation of activators aids or restricts their function.

Mediator acts upstream of the transcriptional activator Gal4.

The proteasome inhibitor MG132 had been shown to prevent galactose induction of the S. cerevisiae GAL1 gene, demonstrating that ubiquitin proteasome-dependent degradation of transcription factors plays an important role in the regulation of gene expression. The deletion of the gene encoding the F-box protein Mdm30 had been reported to stabilize the transcriptional activator Gal4 under inducing conditions and to lead to defects in galactose utilization, suggesting that recycling of Gal4 is required for its function.

Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite.

The recent recognition of Plasmodium falciparum Hsp90 (PfHsp90) as a promising anti-malaria drug target has sparked interest in identifying factors that regulate its function and drug-interaction. Co-chaperones are well-known regulators of Hsp90's chaperone function, and certain members have been implicated in conferring protection against lethal cellular effects of Hsp90-specific inhibitors. In this context, studies on PfHsp90's co-chaperones are imperative to gain insight into the regulation of the chaperone in the malaria parasite.

The histone variant H2A.Z interconverts two stable epigenetic chromatin states.

The nucleosomes occupying the chromosomal start sites of transcription contain the histone H2A variant H2A.Z in place of H2A. Upon galactose induction, nucleosomes are evicted from the GAL1 locus in Saccharomyces cerevisiae cells.

Galactose induction of the GAL1 gene requires conditional degradation of the Mig2 repressor.

Skp1 an essential component of the SCF (Skp1/cullin/F-box) E3 ubiquitin ligases, which target proteins for degradation by the 26S proteasome. We generated a skp1dM mutant strain that is defective for galactose induction of the GAL1 gene and we have found that galactose-induced protein degradation of the repressor Mig2 is defective in this strain. Mig2 degradation was also abolished in cells lacking the protein kinase Snf1 and the F-box protein Das1, suggesting that Snf1 triggers galactose-induced protein degradation of Mig2 by SCFDas1.

Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3.

Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain.

Degradation-resistant protein domains limit host cell processing and immune detection of mycobacteria.

The Mycobacterium tuberculosis genome reveals a large family of glycine-alanine rich PE-PGRS proteins. Due to similarities with the glycine-alanine rich Epstein-Barr nuclear antigen 1, there has been interest in whether PE-PGRS proteins inhibit cellular processing and presentation via the major histocompatibility complex class I pathway. We investigated whether PE-PGRS proteins were resistant to ubiquitin-proteasome-dependent degradation and CD8(+) T cell recognition.

Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II.

Nhp6p is an architectural Saccharomyces cerevisiae non-histone chromosomal protein that bends DNA and plays an important role in transcription and genome stability. We used the split-ubiquitin system to isolate proteins that interact with Nhp6p in vivo, and we confirmed 11 of these protein-protein interactions with glutathione S-transferase pull-down experiments in vitro. Most of the Nhp6p-interacting proteins are involved in transcription and DNA repair.

Gal11p dosage-compensates transcriptional activator deletions via Taf14p.

Transcriptional activators work by recruiting transcription factors that are required for the process of transcription to their target genes. We have used the Split-Ubiquitin system to identify eight transcription factors that interacted with both the transcriptional activators Gal4p and Gcn4p in living cells. The over-expression of one of the activator-interacting proteins, Gal11p, partially suppressed GAL4 and GCN4 deletions.

TFIIB/SUA7(E202G) is an allele-specific suppressor of TBP1(E186D).

The TBP (TATA-box-binding protein), Tbp1p, plays a vital role in all three classes of transcription by RNA polymerases I-III. A TBP1(E186D) mutation had been described that affected interaction of Tbp1p with TFIIB (transcription factor IIB) and that caused slow-growth, temperature-sensitivity, 3-aminotriazole-sensitivity as well as a gal(-) phenotype. We used the TBP1(E186D) mutant for suppressor screens, and we isolated TFIIB/SUA7(E202G) as an allele-specific suppressor of all phenotypes caused by the TBP1(E186D) mutation.

Global effects of histone modifications.

The relationship between chromatin structure and transcriptional regulation has been the focus of many research publications over the past few years. A lot of progress has been made in understanding the role of histone modifications. The interdependence of histone methylation and DNA methylation has been revealed.

Analysis of protein-protein proximities using the split-ubiquitin system.

The split-ubiquitin system is a fragment complementation assay that is based on a conditional proteolysis design. The system has been used to study protein-protein interactions between membrane proteins and to screen for new interaction partners for transcription factors. This paper outlines the recent progress in the split-ubiquitin technology that has been made possible through the development of new reporter proteins.

The cyclin in the RNA polymerase holoenzyme is a target for the transcriptional repressor Tup1p in Saccharomyces cerevisiae.

The general transcriptional repressor Tup1p requires the cyclin/cyclin-dependent kinase pair Srb11p/Srb10p in the holoenzyme of transcription. We used the split-ubiquitin system to demonstrate that Tup1p interacts with Srb11p in vivo. We confirmed our observation in vitro with the help of purified proteins, and we compared the de-repression effect of deleting TUP1, SRB10, and SRB11 on different promoters.

The TATA-binding protein is not an essential target of the transcriptional activators Gal4p and Gcn4p in Saccharomyces cerevisiae.

According to the recruitment model, transcriptional activators work by increasing the local concentration of one or several limiting factors for the transcription process at the target promoter. The TATA-binding protein Tbp1 has been considered as a likely candidate for such a limiting factor. We have used a series of Gal4p and Tbp1 mutants to correlate the in vivo interaction between the two proteins with the strength of activation.

Transcriptional activating regions target a cyclin-dependent kinase.

Several yeast activators are phosphorylated by SRB10, a cyclin-dependent kinase associated with the transcriptional machinery. Sites of phosphorylation are found outside the activating region in each case, and the modification has different physiological consequences in different cases. We show here that certain acidic transcriptional activating regions contact SRB10 as assayed both in vivo and in vitro.