Characterization of Sin1 Isoforms Reveals an mTOR-Dependent and Independent Function of Sin1γ.

Sin1 or MAPKAP1 is a key component of mTORC2 signaling complex which is necessary for AKT phosphorylation at the S473 and T450 sites, and also for AKT downstream signaling as well. A number of Sin1 splicing variants have been reported that can produce different Sin1 isoforms due to exon skipping or alternative transcription initiation. In this report, we characterized four Sin1 isoforms, including a novel Sin1 isoform due to alternative 3' termination of the exon 9a, termed Sin1γ. Sin1γ expression can be detected in multiple adult mouse tissues, and it encodes a C-terminal truncated protein comparing to the full length Sin1β isoform. In contrast to Sin1β, Sin1γ overexpression in Sin1 deficient mouse embryonic fibroblasts has no significant impact on mTORC2 activity or mTORC2 subunits protein level, although it still can interact with mTORC2 components. More interestingly, Sin1γ was detected in a specific cytosolic location with a distinct feature in structure, and its localization was transiently disrupted during cell cycle. Therefore, Sin1γ is a novel Sin1 isoform and may have distinct properties in cell signaling and intracellular localization from other Sin1 isoforms

Knockdown Sin1 protein inhibits cilia formation.

<p>A. The percentage of RPE1 cells with cilia were determined using acetylated-tubulin staining. Average data obtained from two to three different views are shown. Approximately 300 cells for each siRNA transfection were scored each time. Error bar represent SD. *p<0.01 compared with luciferase, #p<0.05 compared with luciferase. B. siRNA knockdown efficiency from (A) were detected using western blot.</p

Characterization of Sin1 isoforms.

<p>A. Schematic map representing of the human Sin1 gene and transcript variants described in this study. There are five Sin1 transcript variants generating four distinct protein isoforms. Two transcript variants encode for Sin1δ. The green and red solid regions represent start and stop codon respectively. B. Sin1 isoforms are widely express in mouse tissues. Total RNA extracted from mice tissues were analyzed by RT-PCR using either Sin1γ specific primers or primers recognized all four isoforms, 18s RNA were used as loading control. Amplified products were separated by electrophoresis in a 2% agarose gel stained with gel red, demonstrating that all four Sin1 isoforms are widely expressed in mice tissues.</p

Sin1γ colocalizes with γ-tubulin.

<p>A. Sin1-/- MEF cells were transiently transfected with indicated GFP-tagged expression plasmid, the slides were fixed, stained with anti-γ-tubulin/Texas red-labelled anti-mouse secondary antibody and analyzed by confocal microscopy after 24h transfection. B. Hela cells were transiently transfected with indicated GFP-tagged expression plasmid, the slides were fixed, stained with anti-γ-tubulin/Texas red-labelled anti-mouse secondary antibody and analyzed by confocal microscopy after 24h transction. Magnification of colocalization between GFP- Sin1γ and γ-tubulin was shown on the right panel. C. Sin1γ expression fluctuates during cell cycle. HeLa cells transfected with GFP empty vector or GFP-Sin1γ for 24h were subjected to time-lapse microscopy observation. Both GFP and DIC channels were shown for cells transfected with each expression plamids. Scale bars: 25 μm (A and C), 5 μm (B). All experiments were repeated for three times with the same results.</p

Restoration of Sin1-/- MEFs with GFP-Sin1 expression vectors.

<p>A. Sin1 isoforms exert distinct functions in restoration of mTORC2 signaling. Sin1-/- MEF cells transfected with GFP empty vector, GFP-Sin1α, GFP-Sin1β, GFP-Sin1γ, GFP-Sin1δ respectively were grown in starved, or starved then restimulated with insulin or serum for 15min. Total cell lysates were analyzed for indicated proteins by immunoblotting. B. The MAPK pathway is not altered in Sin1 isoform-rescued Sin1-/- MEF cells. Sin1-/- MEF cells transfected with GFP empty vector, GFP-Sin1α, GFP-Sin1β, GFP-Sin1γ, GFP-Sin1δ respectively were grown in starved, or starved then restimulated with insulin or serum for 15min. Total cell lysates were analyzed for indicated proteins by immunoblotting. Single-letter abbreviations for the treatments are as follows: S, starvation for 12hr, I, insulin for 15min after starvation, F, FBS for 15min after starvation. C. The quantifications analysis of the phosphor-PKC band are shown.</p

Sin1 isoforms except Sin1δ form mTORC2 complex.

<p>A. Rictor pulled down Sin1α, Sin1β, Sin1γ, but not Sin1δ. HEK-293T cells were transiently transfected for 24h with HA-Sin1 isoform plasmids. Cell lysates (left-hand side) and rictor immunoprecipitates (right-hand side) were analyzed for mTOR, rictor and HA by western blotting. B. Sin1α, Sin1β, Sin1γ, but not Sin1δ could pull down rictor and mTOR. HEK-293T cells were transiently transfected for 24h with HA-Sin1 isoform plasmid respectively. Cell lysates (left-hand side) and HA immunoprecipitates (right-hand side) were analyzed for mTOR, rictor and HA by western blotting. All experiments were repeated for three times with the same results.</p

Keto acid metabolites of branched-chain amino acids inhibit oxidative stress-induced necrosis and attenuate myocardial ischemia–reperfusion injury

Branched chain α-keto acids (BCKAs) are endogenous metabolites of branched-chain amino acids (BCAAs). BCAA and BCKA are significantly elevated in pathologically stressed heart and contribute to chronic pathological remodeling and dysfunction. However, their direct impact on acute cardiac injury is unknown. Here, we demonstrated that elevated BCKAs significantly attenuated ischemia-reperfusion (I/R) injury and preserved post I/R function in isolated mouse hearts. BCKAs protected cardiomyocytes from oxidative stress-induced cell death in vitro. Mechanistically, BCKA protected oxidative stress induced cell death by inhibiting necrosis without affecting apoptosis or autophagy. Furthermore, BCKAs, but not BCAAs, protected mitochondria and energy production from oxidative injury. Finally, administration of BCKAs during reperfusion was sufficient to significantly attenuate cardiac I/R injury. These findings uncover an unexpected role of BCAA metabolites in cardioprotection against acute ischemia/reperfusion injury, and demonstrate the potential use of BCKA treatment to preserve ischemic tissue during reperfusion

Genetic Admixture in the Culturally Unique Peranakan Chinese Population in Southeast Asia

10.1093/molbev/msab187Molecular Biology and Evolution38104463-447

A roadmap for human liver differentiation from pluripotent stem cells

How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah−/−Rag2−/−Il2rg−/− mouse model of liver failure.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Accelerated human liver progenitor generation from pluripotent stem cells by inhibiting formation of unwanted lineages

10.1101/174698BioRxiv17469