Structure and function relationships in the PGC-1 family of transcriptional coactivators

PGC-1 coactivators are central regulators of several cellular processes, most notably the coupling between energy demands and supply. PGCs activate distinct biological processes in a tissue-specific manner. These include mitochondrial biogenesis, oxidative phosphorylation, oxygen transport, gluconeogenesis, angiogenesis, and muscle fiber-type specification. The family consists of three members: PGC-1α, PGC-1β, and PRC, among which the most studied is PGC-1α. Transcription of a single PGC-1α gene produces different isoforms (e.g. PGC-1α1 to α4) with different biological functions. All of the PGC- 1α isoforms share some domain similarity but PGC-1α2, α3 and α4 lack the C-terminal domains present in PGC-1α1. The actions of PGC-1α1 are strongly linked to energy metabolism, whereas PGC-1α4 regulates skeletal muscle hypertrophy. The functions of PGC-1α2 and PGC-1α3 have remained unknown. To study the mechanism of action of the new PGC-1α isoforms, we performed a protein complex purification and identified protein partners of all PGC-1α variants. We found that all PGC-1α isoforms can function as positive regulators of transcription by associating with members of the Mediator complex, histone acetyltransferase complexes, and splicing factors. Furthermore, we identified several transcription factors associated with each PGC- 1α isoform, which allowed us to predict how different target genes are regulated. Interestingly, we also observed that PGC-1α isoforms can regulate splicing events and can affect the exon composition of their corresponding target transcripts. Here, we report for the first time that PGC-1α1 can dimerize with other PGC-1α isoforms, suggesting that some of the functions of PGC-1α might be mediated by heterodimers. Since PGC-1α1 is a key modulator of cellular metabolism in several tissues, it has gained considerable attention as potential target for the treatment of metabolic disorders. For that reason, we developed a screening platform to identify chemicals that can induce PGC-1α1 protein accumulation. From this screen, we identified several candidate small molecules as potential PGC-1α1 activators, which were validated in brown adipocytes. We identified 4 compounds that can increase PGC-1α1 protein accumulation, target gene expression, and mitochondrial respiration. These compounds could represent the beginning of a new class of therapeutics against obesity and related disorders

Skeletal muscle PGC-1α1 reroutes kynurenine metabolism to increase energy efficiency and fatigue-resistance

The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications

The transacting factor CBF-A/Hnrnpab binds to the A2RE/RTS element of protamine 2 mRNA and contributes to its translational regulation during mouse spermatogenesis.

During spermatogenesis, mRNA localization and translation are believed to be regulated in a stage-specific manner. We report here that the Protamine2 (Prm2) mRNA transits through chromatoid bodies of round spermatids and localizes to cytosol of elongating spermatids for translation. The transacting factor CBF-A, also termed Hnrnpab, contributes to temporal regulation of Prm2 translation. We found that CBF-A co-localizes with the Prm2 mRNA during spermatogenesis, directly binding to the A2RE/RTS element in the 3' UTR. Although both p37 and p42 CBF-A isoforms interacted with RTS, they associated with translationally repressed and de-repressed Prm2 mRNA, respectively. Only p42 was found to interact with the 5'cap complex, and to co-sediment with the Prm2 mRNA in polysomes. In CBF-A knockout mice, expression of protamine 2 (PRM2) was reduced and the Prm2 mRNA was prematurely translated in a subset of elongating spermatids. Moreover, a high percentage of sperm from the CBF-A knockout mouse showed abnormal DNA morphology. We suggest that CBF-A plays an important role in spermatogenesis by regulating stage-specific translation of testicular mRNAs

Comparison the prevalence of isolated Hepatitis B core antibody among injection drug users with blood donors in central province in IRAN

Background: In healthy blood donors, 2%–5% have isolated anti-HBc. Prevalence of hepatitis B, C and co- infection (HBV +HCV) among injection drug user is high. Hepatitis C suppress of HB SAg and may be hepatitis B presented only with isolated anti HBC. This study determined of prevalence of isolated anti HBC among injection drug users and compare with blood donors in Arak city. Methods: A total 684 subjects (531voluntary blood donors and 153 injection drug users) in Arak, Iran were included in this study. Hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), anti-HBc, and hepatitis C antibody (anti-HCV) were tested in all subjects. Results: A total of 531 voluntary blood donors living in the city of Arak, in the Central Province of Iran, with a mean age of 36 ± 10.18 years (range 16–60 years) were enrolled in the study. Ninety-three percent of patients were male and 7% were female. Of the 531 cases, 11 subjects (2.1%) had isolated anti-HBc. A total of 153 injection drug users of Arak, in the Central Province of Iran, with a mean age of 30.66 ± 5.92 years (range 20-50 years) were enrolled in the study. All of them were male. Of the 153 cases, 12 subjects (7.84%) had isolated anti-HBc. All of 12 cases were HCV positive. Conclusion: Prevalence of isolated anti-HBc among injection drug users was 3. 73 fold in comparison with blood donors. For diagnosis of hepatitis B in this group test for anti-HBc will be done. Evaluation of occult hepatitis B in subject with isolated anti-HBc by exact method such as real time PCR is necessary

The translationally active <i>Prm2</i> mRNA associates with the p42 CBF-A isoform.

<p>(<b>A–B</b>) Fractionation of adult mouse testicular lysates on a 15–50% continuous sucrose gradient in the absence (A) or presence (B) of EDTA. Fractions were analyzed on Northern blots for the <i>Prm2</i> mRNA and on immunoblots for CBF-A (SAK22) and hnRNP A2. (<b>C–D</b>) The <i>Prm2</i> mRNA is translationally inhibited in postnatal day −28 and −30 mouse testis. (<b>C</b>) Developmental expression of <i>Prm2</i> mRNA in mouse testes. Total RNA was collected from mouse testis of 20, 28, 32 postnatal day of age (20–32 d), and analyzed by RT-PCR. RT, reverse transcriptase. (<b>D</b>) Developmental expression of PRM2 and CBF-A in mouse testes. Cytoplasmic testicular extracts from 20, 28, 30, 32 d mice were analyzed on immunoblots for PRM2, CBF-A (SAK22) and tubulin. (<b>E</b>) RIPs from adult and 30 d mouse testicular extracts. Cytoplasmic fractions were incubated with SAK22, ICCI or control anti-mouse IgGs. Immunoprecipitated fractions were analyzed on immunoblots for CBF-A (SAK22) and hnRNP A2, and by RT-PCR with primers amplifying the <i>Prm2</i>, α-tubulin or clusterin cDNAs. (<b>F–G</b>) The CBF-A p42 isoform associates with the 5′ mRNA cap complex. Cytoplasmic fraction from adult mouse testis was incubated with 7-methyl-GTP (m⁷GTP)-Sepharose or protein G-Sepharose (control). Bound proteins were analyzed on immunoblots for CBF-A (SAK22), hnRNP A2 and eIF4E and α-tubulin. (F) Lanes 1, 2 respectively 2% and 1% input. (G) Lane 1 and 2 are 2% input. Where indicated, testis lysates were pre-incubated with RNase A prior to incubation with the beads.</p

The <i>Prm2</i> mRNA expression is regulated by CBF-A at the translational level during spermatogenesis.

<p>(<b>A</b>) Immunoblots of CBF-A (Sak22), PRM2 and tubulin on Hnrnpab^+/− and Hnrnpab^−/− testis lysates. Total proteins were stained with Coomassie brilliant blue and shown as loading control. Densitometric analysis of the signals in the immunoblots was performed on 3 individual animals per genotypes. Data represent mean +/−SE ^*<i>p</i><0.05 student t-test. (<b>B</b>) Immunostaining for CBF-A on Hnrnpab^+/− and Hnrnpab^−/− testis sections. Bar, 50 µm. (<b>C</b>) Northern blotting analysis for the <i>Prm2</i> mRNA on total RNA from Hnrnpab^+/− and Hnrnpab^−/− mouse testis. Densitometric analysis was on 3 individual animals of each genotype, and shown as mean +/− SE. (<b>D</b>) <i>In situ</i> hybridization on Hnrnpab^−/− and Hnrnpab^+/− testis sections showed no differences on <i>Prm2</i> mRNA expression. Bar, 50 µm. The inset shows a magnified image of the marked area. (<b>E</b>) Fractionation of testicular lysates from adult Hnrnpab^+/+ and Hnrnpab^−/− mice on a 15–50% continuous sucrose gradient. Fractions were analyzed on Northern blots for the <i>Prm2</i> mRNA. (<b>F</b>) Testis section of Hnrnpab^+/− stained with a PRM2 antibody (Green) and DAPI (gray). PRM2 was not detected in the seminiferous tubules of stage IX–XII, in which pachytene spermatocytes (P) and elongating spermatids (EL) are contained. (<b>G</b>) Testis section from Hnrnpab^−/− stained with a PRM2 antibody (Green) and DAPI (gray). PRM2 signal was detected in some nuclei of elongating spermatids. (<b>H and I</b>) Magnified image of marked area in (G). DAPI staining shows that chromatin of PRM2-positive nuclei (white arrow head) appears more condensed than in PRM2-negative nuclei (blank arrow head). (<b>J</b>) Testis section of Hnrnpab^−/− stained with a PRM2 antibody (Green) and DAPI (gray). The seminiferous tubule was categorized as stage IX–XII, because it does not contain round-spermatid layer (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858.s007" target="_blank">Figure S7</a>), but nuclei of elongating spermatids appear prematurely condensed and positive for PRM2 signals. * denotes background staining of Leydig cells with the PRM2 antibody. Bars, 50 µm.</p

Immunostaining on tubule squash preparations of testicular cells at different steps of spermatogenesis.

<p>Cells were triple-stained with the antisense probe for the <i>Prm2</i> mRNA (red), an anti-PRM2 antibody (green) and with the anti-MVH antibody (white) as well as DAPI. The <i>Prm2</i> mRNA was co-localized with MVH to chromatoid bodies as from step 7–8 spermatids. In late step spermatids (step 9–12 and step 13–15), the <i>Prm2</i> mRNA becomes more diffusely localized to the cytosol. Scale bars, 10 µm (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858.s003" target="_blank">Figure S3</a> for more examples).</p

<i>In vivo</i> localization of <i>Prm2</i> mRNA in adult mouse testes.

<p>(<b>A</b>) <i>In situ</i> hybridization on testis cryosections (10 µm) using a <i>Prm2</i> mRNA antisense probe. Nuclei were stained with DAPI and shown in blue. Roman numbers indicate stages of seminiferous epithelium cycle. (<b>B</b>) Diagram of the 12-stage growth cycle of mouse spermatogenesis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858-Russell1" target="_blank">[55]</a>, showing the stages of the <i>Prm2</i> mRNA expression during spermatogenesis. The <i>Prm2</i> mRNA is detected from step 7 round spermatids to step 14 elongated spermatids (highlighted in magenta). (<b>C</b>) Subcellular localization of the <i>Prm2</i> mRNA in spermatids during spermatogenesis. Testis cryosections were double stained with the <i>Prm2</i> mRNA antisense probe (red, upper panels) and the anti-MVH antibody (white, lower panels). The <i>Prm2</i> mRNA was localized to perinuclear structures positive for the chromatoid body marker MVH in the spermatids at stage VII–VIII and dispersed into cytosol at later stages. Scale bar, A; 100 µm, C; 20 µm.</p

CBF-A is in cytoplasmic RNPs in complex with the <i>Prm2</i> mRNA in testicular cells.

<p>(<b>A</b>) Immunoblots of nuclear and cytoplasmic fractions of testis lysates. Anti-fibrillarin (nucleolar protein) and anti-Tom20 (mitochondrial protein) antibodies were used to characterize the fractionation. (<b>B</b>) Untreated or RNase-treated cytoplasmic fractions of testis lysates were incubated with anti-CBF-A antibodies (SAK22 or ICCI) or control non-specific IgGs and the immunoprecipitates were probed as indicated. * marks a testis-specific variant of hnRNP A2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858-Kamma1" target="_blank">[56]</a>. (<b>C</b>) RNA immunoprecipitation (RIP) assays. Cytoplasmic fractions of testis lysates were incubated with anti-CBF-A antibodies (SAK22 or ICCI), control anti-mouse IgGs, or without antibodies (mock). In all cases, total RNA was extracted from the immunoprecipitates and analyzed by RT-PCR with primers amplifying <i>Prm2</i>, α-tubulin or clusterin cDNA. (<b>D</b>) Densitometric quantifications of the RIP experiments. The signal intensities of the RT-PCR bands were calculated from 3 independent experiments and shown as % of input (mean+/−SE).</p

Transmission electron micrographs of sperm heads from the epididymis from Hnrnpab^−/− and Hnrnpab^+/− mice.

<p>(A) Normal sperm from Hnrnpab^−/− mouse showing the nucleus (n) revealed like an evenly stained black mass (due to high electron density) that contains the highly compacted DNA, surrounded by the nuclear envelope. The acrosome (a) located in the anterior half of the head and the proximal centriole (pc) in the neck of the sperm. (B) Abnormal sperm of Hnrnpab^−/− mice with fibers extending out of the main mass of local regions of nucleus (inset in B and B′). (C) Abnormal sperm of Hnrnpab^−/− mice with fibers extending out of the main mass around the nucleus (inset in C and C′). (D) Elongated spermatid at step 13 of spermatogenesis of wild-type mice. Posterior part of DNA, which is less packed than in anterior region, was observed as fibers (inbox in D and D′). Scale bar is 2 µm in A–D and 500 nm in B′–D′.</p