Randy Strich

Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1.

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding-induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis.

Stress and developmental regulation of the yeast C‐type cyclin Ume3p (Srb11p/Ssn8p)

The ume3‐1 allele was identified as a mutation that allowed the aberrant expression of several meiotic genes (e.g. SPO11, SPO13) during mitotic cell division in Saccharomyces cerevisiae. Here we report that UME3 is also required for the full repression of the HSP70 family member SSA1. UME3 encodes a non‐essential C‐type cyclin (Ume3p) whose levels do not vary through the mitotic cell cycle. However, Ume3p is destroyed during meiosis or when cultures are subjected to heat shock. Ume3p mutants resistant to degradation resulted in a 2‐fold reduction in SPO13 mRNA levels during meiosis, indicating that the down‐regulation of this cyclin is important for normal meiotic gene expression. Mutational analysis identified two regions (PEST‐rich and RXXL) that mediate Ume3p degradation. A third destruction signal lies within the highly conserved cyclin box, a region that mediates cyclin–cyclin‐dependent kinase (Cdk) interactions. However, the Cdk activated by Ume3p (Ume5p) is not required for the rapid destruction of this cyclin. Finally, Ume3p destruction was not affected in mutants defective for ubiquitin‐dependent proteolysis. These results support a model in which Ume3p, when exposed to heat shock or sporulation conditions, is targeted for destruction to allow the expression of genes necessary for the cell to respond correctly to these environmental cues.

Oxidative Stress-Induced Destruction of the Yeast C-Type Cyclin Ume3p Requires Phosphatidylinositol-Specific Phospholipase C and the 26S Proteasome

ABSTRACT The yeast UME3 (SRB11/SSN3) gene encodes a C-type cyclin that represses the transcription of the HSP70family member SSA1. To relieve this repression, Ume3p is rapidly destroyed in cells exposed to elevated temperatures. This report demonstrates that Ume3p levels are also reduced in cultures subjected to ethanol shock, oxidative stress, or carbon starvation or during growth on nonfermentable carbons. Of the three elements (RXXL, PEST, and cyclin box) previously shown to be required for heat-induced Ume3p destruction, only the cyclin box regulates Ume3p degradation in response to these stressors. The one exception observed was growth on nonfermentable carbons, which requires the PEST region. These findings indicate that yeast cells contain multiple, independent pathways that mediate stress-induced Ume3p degradation. Ume3p destruction in response to oxidative stress, but not to ethanol treatment, requires DOA4 and UMP1, two factors required for 26S proteasome activity. This result for the first time implicates ubiquitin-mediated proteolysis in C-type cyclin regulation. Similarly, the presence of a membrane stabilizer (sorbitol) or the loss of phosphatidylinositol-specific phospholipase C (PLC1) protects Ume3p from oxidative-stress-induced degradation. Finally, a ume3 null allele suppresses the growth defect of plc1 mutants in response to either elevated temperature or the presence of hydrogen peroxide. These results indicate that the growth defects observed in plc1mutants are due to the failure to downregulate Ume3p. Taken together, these findings support a model in which Plc1p mediates an oxidative-stress signal from the plasma membrane that triggers Ume3p destruction through a Doa4p-dependent mechanism.

In vitro biodegradation behavior, mechanical properties, and cytotoxicity of biodegradable Zn–Mg alloy

Zinc-Magnesium (Zn-Mg) alloy as a novel biodegradable metal holds great potential in biodegradable implant applications as it is more corrosion resistant than Magnesium (Mg). However, the mechanical properties, biodegradation uniformity, and cytotoxicity of Zn-Mg alloy remained as concerns. In this study, hot extrusion process was applied to Zn-1 wt % Mg (Zn-1Mg) to refine its microstructure. Effects of hot extrusion on biodegradation behavior and mechanical properties of Zn-1Mg were investigated in comparison with Mg rare earth element alloy WE43. Metallurgical analysis revealed significant grain size reduction, and immersion test found that corrosion rates of WE43 and Zn-1Mg were reduced by 35% and 57%, respectively after extrusion. Moreover, hot extrusion resulted in a much more uniform biodegradation in extruded Zn-1Mg alloy and WE43. In vitro cytotoxicity test results indicated that Zn-1Mg alloy was biocompatible. Therefore, hot extruded Zn-1Mg with homogenous microstructure, uniform as well as slow degradation, improved mechanical properties, and good biocompatibility was believed to be an excellent candidate material for load-bearing biodegradable implant application.

Ask10p mediates the oxidative stress-induced destruction of the Saccharomyces cerevisiae C-type cyclin Ume3p/Srb11p.

ABSTRACT Srb11p-Srb10p is the budding yeast C-type cyclin-cyclin-dependent kinase that is required for the repression of several stress response genes. To relieve this repression, Srb11p is destroyed in cells exposed to stressors, including heat shock and oxidative stress. In the present study, we identified Ask10p (for activator of Skn7) by two-hybrid analysis as an interactor with Srb11p. Coimmunoprecipitation studies confirmed this association, and we found that, similar to Srb11p-Srb10p, Ask10p is a component of the RNA polymerase II holoenzyme. Ask10p is required for Srb11p destruction in response to oxidative stress but not heat shock. Moreover, this destruction is important since the hypersensitivity of an ask10 mutant strain to oxidative stress is rescued by deleting SRB11. We further show that Ask10p is phosphorylated in response to oxidative stress but not heat shock. This modification requires the redundant mitogen-activated protein (MAP) kinase kinase Mkk1/2 but not their normal MAP kinase target Slt2p. Moreover, the other vegetative MAP kinases—Hog1p, Fus3p, or Kss1p—are not required for Ask10p phosphorylation, suggesting the existence of an alternative pathway for transducing the Pkc1p→Bck1→Mkk1/2 oxidative stress signal. In conclusion, Ask10p is a new component of the RNA polymerase II holoenzyme and an important regulator of the oxidative stress response. In addition, these results define a new role for the Pkc1p MAP kinase cascade (except the MAP kinase itself) in transducing the oxidative damage signal directly to the RNA polymerase II holoenzyme, thereby bypassing the stress-activated transcription factors.

Stress-induced nuclear-to-cytoplasmic translocation of cyclin C promotes mitochondrial fission in yeast.

Mitochondrial morphology is maintained by the opposing activities of dynamin-based fission and fusion machines. In response to stress, this balance is dramatically shifted toward fission. This study reveals that the yeast transcriptional repressor cyclin C is both necessary and sufficient for stress-induced hyperfission. In response to oxidative stress, cyclin C translocates from the nucleus to the cytoplasm, where it is destroyed. Prior to its destruction, cyclin C both genetically and physically interacts with Mdv1p, an adaptor that links the GTPase Dnm1p to the mitochondrial receptor Fis1p. Cyclin C is required for stress-induced Mdv1p mitochondrial recruitment and the efficient formation of functional Dnm1p filaments. Finally, coimmunoprecipitation studies and fluorescence microscopy revealed an elevated association between Mdv1p and Dnm1p in stressed cells that is dependent on cyclin C. This study provides a mechanism by which stress-induced gene induction and mitochondrial fission are coordinated through translocation of cyclin C.

Oxidative-stress-induced nuclear to cytoplasmic relocalization is required for Not4-dependent cyclin C destruction

The yeast cyclin-C–Cdk8p kinase complex represses the transcription of a subset of genes involved in the stress response. To relieve this repression, cyclin C is destroyed in cells exposed to H2O2 by the 26S proteasome. This report identifies Not4p as the ubiquitin ligase mediating H2O2-induced cyclin C destruction. Not4p is required for H2O2-induced cyclin C destruction in vivo and polyubiquitylates cyclin C in vitro by utilizing Lys48, a ubiquitin linkage associated with directing substrates to the 26S proteasome. Before its degradation, cyclin C, but not Cdk8p, translocates from the nucleus to the cytoplasm. This translocation requires both the cell-wall-integrity MAPK module and phospholipase C, and these signaling pathways are also required for cyclin C destruction. In addition, blocking cytoplasmic translocation slows the mRNA induction kinetics of two stress response genes repressed by cyclin C. Finally, a cyclin C derivative restricted to the cytoplasm is still subject to Not4p-dependent destruction, indicating that the degradation signal does not occur in the nucleus. These results identify a stress-induced proteolytic pathway regulating cyclin C that requires nuclear to cytoplasmic relocalization and Not4p-mediated ubiquitylation.

Saccharomyces cerevisiae C-type cyclin Ume3p/Srb11p is required for efficient induction and execution of meiotic development.

ABSTRACT The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase partner Ume5p/Srb10p repress the transcription of several genes required for meiotic recombination or meiosis I nuclear division. To relieve this repression, Srb11p is destroyed early in meiosis, prior to the first meiotic division. This report identifies two roles for Srb11p in regulating meiotic development. First, SRB11 is required for the normal exit from the mitotic cell cycle prior to meiotic induction. Specifically, mutants lacking SRB11 (srb11Δ) uncouple bud growth from chromosome segregation, producing small buds with nuclei. The bud growth defect is most likely due to the failure of srb11Δ mutants to reestablish polarized actin fibers at the bud tip following exposure to sporulation medium. Second, Srb11p is required for the efficient execution of meiosis I. srb11Δ mutants either exhibited a delay in performing meiosis I and meiosis II or skipped meiosis I entirely. This meiotic defect is not due to the activation of the recombination or spindle assembly checkpoint pathways. However, the expression of several meiotic genes is delayed and reduced in the mutant strains. These results suggest a positive role for Srb10-Srb11p in regulating the transcription program. This model is supported by the finding that overexpression of the meiotic inducer IME2 partially restored the ability of srb11 mutants to perform meiosis I. In conclusion, these findings indicate that Srb11p is required for both entry into and execution of the meiotic program, thus describing multiple roles for a C-type cyclin in the regulation of a developmental pathway.

Meiotic control of the APC/C: similarities & differences from mitosis

The anaphase promoting complex is a highly conserved E3 ligase complex that mediates the destruction of key regulatory proteins during both mitotic and meiotic divisions. In order to maintain ploidy, this destruction must occur after the regulatory proteins have executed their function. Thus, the regulation of APC/C activity itself is critical for maintaining ploidy during all types of cell divisions. During mitotic cell division, two conserved activator proteins called Cdc20 and Cdh1 are required for both APC/C activation and substrate selection. However, significantly less is known about how these proteins regulate APC/C activity during the specialized meiotic nuclear divisions. In addition, both budding yeast and flies utilize a third meiosis-specific activator. In Saccharomyces cerevisiae, this meiosis-specific activator is called Ama1. This review summarizes our knowledge of how Cdc20 and Ama1 coordinate APC/C activity to regulate the meiotic nuclear divisions in yeast.

Meiotic DNA Replication

Abstract Meiosis is the process by which diploid organisms produce haploid gametes capable of sexual reproduction. During meiosis, the cell performs one round of DNA replication (meiS) followed by homolog synapsis and extensive genetic recombination. Haploidization is then achieved through two subsequent nuclear divisions (meiosis I and meiosis II) without an intervening S phase. Several recent studies have found that unique properties of meiS are required to prepare the chromosomes for genetic recombination and the reductional meiosis I nuclear division. Although much of the basic replication apparatus is employed to perform meiS, studies from the budding and fission yeasts have also uncovered meiosis-specific regulators that usurp the mitotic cell cycle machinery to perform these specialized landmark events. This “same gun, different trigger” approach is employed for the initiation of DNA replication, recombination, and the meiosis I nuclear division. In addition to the correct induction and execution of meiS, this process must be precisely regulated. Not only must meiS be restricted to once and only once during the normal replication window, it must also be prevented between the two meiotic nuclear divisions. Current data indicate that blocking rereplication between meiosis I and meiosis II uses a strategy similar to mitotic cell division. However, the block to rereplication during the normal meiS window appears to apply a very different approach to solve this problem.