Insights into the function of HDAC3 and NCoR1/NCoR2 co-repressor complex in metabolic diseases.

Histone deacetylase 3 (HDAC3) and nuclear receptor co-repressor (NCoR1/2) are epigenetic regulators that play a key role in gene expression and metabolism. HDAC3 is a class I histone deacetylase that functions as a transcriptional co-repressor, modulating gene expression by removing acetyl groups from histones and non-histone proteins. NCoR1, on the other hand, is a transcriptional co-repressor that interacts with nuclear hormone receptors, including peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR), to regulate metabolic gene expression.

The lipid droplet-associated protein ABHD5 protects the heart through proteolysis of HDAC4.

Catecholamines stimulate the first step of lipolysis by PKA-dependent release of the lipid droplet-associated protein ABHD5 from perilipin to co-activate the lipase ATGL. Here, we unmask a yet unrecognized proteolytic and cardioprotective function of ABHD5. ABHD5 acts and as a serine protease cleaving HDAC4.

The Insoluble Protein Deposit (IPOD) in Yeast.

The appearance of protein aggregates is a hallmark of several pathologies including many neurodegenerative diseases. Mounting evidence suggests that the accumulation of misfolded proteins into inclusions is a secondary line of defense when the extent of protein misfolding exceeds the capacity of the Protein Quality Control System, which mediates refolding or degradation of misfolded species. Such exhaustion can occur during severe proteotoxic stress, the excessive occurrence of aggregation prone protein species, e.

Effects of the Reactive Metabolite Methylglyoxal on Cellular Signalling, Insulin Action and Metabolism - What We Know in Mammals and What We Can Learn From Yeast.

Levels of reactive metabolites such as reactive carbonyl and oxygen species are increased in patients with diabetes mellitus. The most important reactive dicarbonyl species, methylglyoxal (MG), formed as by-product during glucose metabolism, is more and more recognized as a trigger for the development and progression of diabetic complications. Although it is clear that MG provokes toxic effects, it is currently not well understood what cellular changes MG induces on a molecular level that may lead to pathophysiological conditions found in long-term diabetic complications.

Hormesis enables cells to handle accumulating toxic metabolites during increased energy flux.

Energy production is inevitably linked to the generation of toxic metabolites, such as reactive oxygen and carbonyl species, known as major contributors to ageing and degenerative diseases. It remains unclear how cells can adapt to elevated energy flux accompanied by accumulating harmful by-products without taking any damage. Therefore, effects of a sudden rise in glucose concentrations were studied in yeast cells.

Hitchhiking vesicular transport routes to the vacuole: Amyloid recruitment to the Insoluble Protein Deposit (IPOD).

Sequestration of aggregates into specialized deposition sites occurs in many species across all kingdoms of life ranging from bacteria to mammals and is commonly believed to have a cytoprotective function. Yeast cells possess at least 3 different spatially separated deposition sites, one of which is termed "Insoluble Protein Deposit (IPOD)" and harbors amyloid aggregates. We have recently discovered that recruitment of amyloid aggregates to the IPOD uses an actin cable based recruitment machinery that also involves vesicular transport.

Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD) via Myosin 2-Based Vesicular Transport.

Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed "Insoluble PrOtein Deposit (IPOD)", but nothing is known about the mechanism of substrate recruitment to this site.

Characterization of aggregate load and pattern in living yeast cells by flow cytometry.

Protein aggregation is both a hallmark of and a driving force for a number of diseases. It is therefore important to identify the nature of these aggregates and the mechanism(s) by which the cell counteracts their detrimental properties. Currently, the study of aggregation in vivo is performed primarily using fluorescently tagged versions of proteins and analyzing the aggregates by fluorescence microscopy.

Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae.

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae.

daf-16/FOXO and glod-4/glyoxalase-1 are required for the life-prolonging effect of human insulin under high glucose conditions in Caenorhabditis elegans.

Aims/hypothesis: The aim of this study was to determine the protective effects of human insulin and its analogues, B28Asp human insulin (insulin aspart) and B29Lys(ε-tetradecanoyl),desB30 human insulin (insulin detemir), against glucose-induced lifespan reduction and neuronal damage in the model organism Caenorhabditis elegans and to elucidate the underlying mechanisms.

Methods: Nematodes were cultivated under high glucose (HG) conditions comparable with the situation in diabetic patients and treated with human insulin and its analogues. Lifespan was assessed and neuronal damage was evaluated with regard to structural and functional impairment.

Monitoring protein misfolding by site-specific labeling of proteins in vivo.

Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [PSI+] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase in vivo.

Coordination of translational control and protein homeostasis during severe heat stress.

Background: Exposure of cells to severe heat stress causes not only misfolding and aggregation of proteins but also inhibition of translation and storage of mRNA in cytosolic heat stress granules (heat-SGs), limiting newly synthesized protein influx into overloaded proteome repair systems. How these two heat stress responses connect is unclear.

Results: Here, we show that both S.

Aging-dependent reduction in glyoxalase 1 delays wound healing.

Methylglyoxal (MG), the major dicarbonyl substrate of the enzyme glyoxalase 1 (GLO1), is a reactive metabolite formed via glycolytic flux. Decreased GLO1 activity in situ has been shown to result in an accumulation of MG and increased formation of advanced glycation endproducts, both of which can accumulate during physiological aging and at an accelerated rate in diabetes and other chronic degenerative diseases. To determine the physiological consequences which result from elevated MG levels and the role of MG and GLO1 in aging, wound healing in young (≤12 weeks) and old (≥52 weeks) wild-type mice was studied.

Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures.

Yeast prions constitute a "protein-only" mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI(+)], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits.

Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation.

Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA(+) hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial.

Chaperone networks in protein disaggregation and prion propagation.

The oligomeric AAA+ chaperones Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 cooperate with cognate Hsp70/Hsp40 chaperone machineries in the reactivation of aggregated proteins in E. coli and S. cerevisiae.

Patterns of [PSI (+) ] aggregation allow insights into cellular organization of yeast prion aggregates.

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI (+) ] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD).

Prion formation and polyglutamine aggregation are controlled by two classes of genes.

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN+].

Cellular strategies for controlling protein aggregation.

The aggregation of misfolded proteins is associated with the perturbation of cellular function, ageing and various human disorders. Mounting evidence suggests that protein aggregation is often part of the cellular response to an imbalanced protein homeostasis rather than an unspecific and uncontrolled dead-end pathway. It is a regulated process in cells from bacteria to humans, leading to the deposition of aggregates at specific sites.

Prion induction involves an ancient system for the sequestration of aggregated proteins and heritable changes in prion fragmentation.

When the translation termination factor Sup35 adopts the prion state, [PSI(+)], the read-through of stop codons increases, uncovering hidden genetic variation and giving rise to new, often beneficial, phenotypes. Evidence suggests that prion induction involves a process of maturation, but this has never been studied in detail. To do so, we used a visually tractable prion model consisting of the Sup35 prion domain fused to GFP (PrD-GFP) and overexpressed it to achieve induction in many cells simultaneously.

Prion switching in response to environmental stress.

Evolution depends on the manner in which genetic variation is translated into new phenotypes. There has been much debate about whether organisms might have specific mechanisms for "evolvability," which would generate heritable phenotypic variation with adaptive value and could act to enhance the rate of evolution. Capacitor systems, which allow the accumulation of cryptic genetic variation and release it under stressful conditions, might provide such a mechanism.

The Sec61p complex is a dynamic precursor activated channel.

Previous studies have shown that the rough endoplasmic reticulum (ER) contains nascent precursor polypeptide gated channels. Circumstantial evidence suggests that these channels are formed by the Sec61p complex. We reconstituted the purified Sec61p complex in a lipid bilayer and characterized its dynamics and regulation.

Polypeptide-binding proteins mediate completion of co-translational protein translocation into the mammalian endoplasmic reticulum.

The first step in the secretion of most mammalian proteins is their transport into the lumen of the endoplasmic reticulum (ER). Transport of pre-secretory proteins into the mammalian ER requires signal peptides in the precursor proteins and a protein translocase in the ER membrane. In addition, hitherto unidentified lumenal ER proteins have been shown to be required for vectorial protein translocation.

Tail-anchored and signal-anchored proteins utilize overlapping pathways during membrane insertion.

Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins.