Differential Localization of G Protein βγ Subunits

G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system

Yeast require redox switching in DNA primase

Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]^+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo

The Anaphase Promoting Complex Contributes to the Degradation of the S. cerevisiae Telomerase Recruitment Subunit Est1p

Telomerase is a multi-subunit enzyme that reverse transcribes telomere repeats onto the ends of linear eukaryotic chromosomes and is therefore critical for genome stability. S. cerevisiae telomerase activity is cell-cycle regulated; telomeres are not elongated during G1 phase. Previous work has shown that Est1 protein levels are low during G1 phase, preventing telomerase complex assembly. However, the pathway targeting Est1p for degradation remained uncharacterized. Here, we show that Est1p stability through the cell cycle mirrors that of Clb2p, a known target of the Anaphase Promoting Complex (APC). Indeed, Est1p is stabilized by mutations in both essential and non-essential components of the APC. Mutations of putative Destruction boxes (D-boxes), regions shown to be important for recognition of known APC substrates, stabilize Est1p, suggesting that Est1p is likely to be targeted for degradation directly by the APC. However, we do not detect degradation or ubiquitination of recombinant Est1p by the APC in vitro, suggesting either that the recombinant protein lacks necessary post-translational modification and/or conformation, or that the APC affects Est1p degradation by an indirect mechanism. Together, these studies shed light on the regulation of yeast telomerase assembly and demonstrate a new connection between telomere maintenance and cell cycle regulation pathways

Point of View: What’s in a name?

Numerous concerns have been raised about the sustainability of the biomedical research enterprise in the United States. Improving the postdoctoral training experience is seen as a priority in addressing these concerns, but even identifying who the postdocs are is made difficult by the multitude of different job titles they can carry. Here, we summarize the detrimental effects that current employment structures have on training, compensation and benefits for postdocs, and argue that academic research institutions should standardize the categorization and treatment of postdocs. We also present brief case studies of two institutions that have addressed these challenges and can provide models for other institutions attempting to enhance their postdoctoral workforces and improve the sustainability of the biomedical research enterprise

Yeast require redox switching in DNA primase

Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]^+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo

Fetal Epidermal Differentiation and Barrier Development In Vivo is Accelerated by Nuclear Hormone Receptor Activators1

Nuclear receptors which interact with the retinoid X receptor are involved in the regulation of epidermal differentiation and development. We have recently shown that activators of the peroxisome proliferator-activated receptor and of the farnesoid X-activated receptor accelerate epidermal barrier maturation in fetal rat skin in vitro. In this study we asked whether cutaneous development in utero was affected by peroxisome proliferator-activated receptor or farnesoid X-activated receptor activators, or by an activator of another retinoid X receptor partner, liver X receptor. Activators of the peroxisome proliferator-activated receptor (clofibrate or linoleic acid), farnesoid X-activated receptor (farnesol or juvenile hormone III), or liver X receptor (22R-hydroxycholesterol), were injected into the amniotic fluid of fetal rats on gestational day 17. Fetal epidermal barrier function and morphology was assessed on day 19. Whereas vehicle-treated fetal rats displayed no measurable barrier (transepidermal water loss > 10 mg per cm2 per h), a measurable barrier was induced by the intra-amniotic administration of all activators tested (transepidermal water loss range 4.0–8.5 mg per cm2 per h). By light microscopy, control pups lacked a well-defined stratum corneum, whereas a distinct stratum corneum and a thickened stratum granulosum were present in treated pups. By electron microscopy, the extracellular spaces of the stratum corneum in control pups revealed a paucity of mature lamellar unit structures, whereas these structures filled the stratum corneum interstices in treated pups. Additionally, protein and mRNA levels of loricrin and filaggrin, two structural proteins of stratum corneum, were increased in treated epidermis, as were the activities of two lipid catabolic enzymes critical to stratum corneum function, β-glucocerebrosidase and steroid sulfatase. Finally, peroxisome proliferator-activated receptor-α and -δ and liver X receptor-α and -β mRNAs were detected in fetal epidermis by reverse transcriptase–polymerase chain reaction and northern analyses. The presence of these receptors and the ability of their activators to stimulate epidermal barrier and stratum corneum development suggest a physiologic role for peroxisome proliferator-activated receptor and liver X receptor and their endogenous ligands in the regulation of cutaneous development

Participation in Cancer Pharmacogenomic Studies: A Study of 8456 Patients Registered to Clinical Trials in the Cancer and Leukemia Group B (Alliance)

Clinically annotated specimens from cancer clinical trial participants offer an opportunity for discovery and validation of pharmacogenomic findings. The purpose of this observational study is to better understand patient/institution factors that may contribute to participation in the pharmacogenomic component of prospective cancer clinical trials

Differential expression of transforming growth factor-β isoforms and receptors in experimental membranous nephropathy

Transforming growth factor-β1 stimulates matrix production by glomerular mesangial and epithelial cells. In membranous nephropathy (MN) overproduction of matrix by glomerular epithelial cells (GEC) is believed to be responsible for glomerular basement membrane thickening and spikes. We studied experimental MN in rats (passive Heymann nephritis, PHN) at 5, 10 and 30 days. PHN rats exhibited a marked increase in GEC immunostaining for TGF-β2 at all time points. TGF-β3 staining was increased at day 10 only, and TGF-β1 was unchanged. Glomerular mRNA for TGF-β2 and -β3 was increased by day 5 when urine protein increased, whereas TGF-β1 was not. TGF-β2 bioactivity was increased at day 5. There was also a marked increase in GEC immunostaining for TGF-β receptor type I (TβIR) and TGF-β receptor type II (TβIIR) at all time points in PHN. mRNA levels for both receptors increased at day 5. Increases in protein expression and mRNA levels for the TGF-β2 and -β3 isoforms, and TβIR and TβRII were prevented by complement depletion. We conclude that complement-mediated injury to the GEC in vivo is associated with the up-regulation of TGF-β2 and -β3 isoforms, an increase in TGF-β2 bioactivity, and an increase in TβRI and TβRII expression. This contrasts with changes in TGF-β1 reported in mesangial disease, suggesting that TGF-β2 and -β3 may be important in diseases of the GEC. The differential expression of TGF-β isoforms and receptors may be important determinants of the GEC response to injury