Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells

Autologous c-kit⁺ cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G₁ phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G₂/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.     

Molecular Phylogeny,Evolution, and Functional Divergence of the LSD1-Like Gene Family: Inference from the Rice Genome

The identification of LSD1-like genes in parasite, green algae, moss, pine, and monocot and dicot species allowed us to trace the phylogenetic history of this gene family. Computational analysis showed that the diversification of members of this family could be dated back to the early stage of plant evolution. The evolution of plant LSD1-like genes was possibly shaped by two duplication events. These proteins, which contain three copies of the LSD1 zinc finger (zf-LSD1) domain within their entire polypeptides and play crucial roles in modulating disease defense and cell death, resulted from the second duplication. A gain of zf-LSD1 domain model was reasonable for explaining the origination of three-zf-LSD1 domain-containing proteins. The zf-LSD1 domain phylogeny showed that the middle (M) and C-terminal (C) domains originated from a common ancestor; the N-terminal (N) domain might be more ancient than the former two. The divergence of the N, M, and C domains was well before the monocot-dicot split. Coevolution analysis revealed that four intramolecular domain pairs, including the N domain and the interregion between the M and the C domains (INTER2), the M and C domain, the N- and C-terminus, and the M domain and C-terminus, possibly coevolved during the evolution of three-zf-LSD1 domain-containing proteins. The three zf-LSD1 domains are evolutionary conserved. Thus, the differences at the N- and C-terminus would be crucial for functional specificity of LSD1 genes. Strong functional constraints should work on the zf-LSD1 domains, whereas reduced functional constraint was found in the INTER2 region. Functional divergence analysis showed that three-zf-LSD1 domain-containing proteins were significantly functionally divergent from those proteins containing only one zf-LSD1 domain, a result demonstrating that shifted evolutionary rates between the two clusters were significantly different from each other. [Reviewing Editor: Dr. Joshua Plotkin]     

Expression of Musashi-1 in the rat stomach and changes during mucosal injury and restitution

Musashi-1 is involved in maintenance of neural stem cells. In the rat stomach, we found some cells in the luminal compartment of the mucosa that stained positively for Musashi-1. These cells were distinct from other cells of epithelial lineage, except for parietal cells, and coexpressed HES5. The Musashi-1-positive cells exfoliated after damage, while Musashi-1 expression in neck cells were upregulated, and proliferating cells diminished before reappearing and increasing in number thereafter. We conclude that a subpopulation of parietal cells acts as a source of Musashi-1, which contributes to rapid re-epithelization by restoration of stem cells and regulation of cell differentiation.     

Epigenetic upregulation and functional role of the mitochondrial aspartate/glutamate carrier isoform 1 in hepatocellular carcinoma

Metabolic reprogramming is a common hallmark of cancer cells. Although some biochemical features have been clarified, there is still much to learn about cancer cell metabolism and its regulation. Aspartate-glutamate carrier isoform 1 (AGC1), encoded by SLC25A12 gene, catalyzes an exchange between intramitochondrial aspartate and cytosolic glutamate plus a proton across the mitochondrial membrane, so supplying aspartate to the cytosol. SLC25A12, expressed in brain, heart, and skeletal muscle, is silenced in normal liver. Here, we demonstrate that SLC25A12 gene is reactivated in hepatocellular carcinoma (HCC) HepG2 cell line through histone acetylation and CREB recruitment. Furthermore, SLC25A12 knockdown by small interfering RNA, impairs HepG2 cell proliferation by inducing cell cycle arrest. AGC1 sustains HCC cell growth by supplying cytosolic aspartate for nucleotide biosynthesis. In addition, SLC25A12-silenced HCC cells show a strong reduction of cell migration. Overall, we have provided evidence for molecular mechanisms controlling SLC25A12 gene expression in liver and pointing to an important role for AGC1 in HCC.     

Functional Analysis of the Relationship between Vpx and the Restriction Factor SAMHD1

SAMHD1 is a newly identified restriction factor that targets lentiviruses in myeloid cells and is countered by the SIV_SM/HIV-2 Vpx protein. By analyzing a large panel of Vpx mutants, we identify several residues throughout the 3-helix bundle predicted for Vpx that impair both its functionality and its ability to degrade SAMHD1. We determine that SAMHD1 is a strictly non-shuttling nuclear protein and that as expected WT Vpx localizes with it in the nucleus. However, we also identify a functional Vpx mutant with predominant cytoplasmic distribution that colocalizes with SAMHD1 in this location, suggesting that Vpx may also retain SAMHD1 in the cell cytoplasm, prior to its entry into the nucleus. Several mutations in Vpx were shown to affect the stability of Vpx, as well as Vpx:Vpx interactions. However, no strict correlation was observed between these parameters and the functionality of Vpx, implying that neither properties is absolutely required for this function and indicating that even unstable Vpx mutants may be very efficient in inducing SAMHD1 degradation. Overall, our analysis identifies several Vpx residues required for SAMHD1 degradation and points to a very efficient and plastic mechanism through which Vpx depletes this restriction factor.     

Structural and Functional Analyses of the Hfb1, Hmob3, and Hmob33 cDNAs as an Example of Human Brain-Specific Gene Studies

Brain-specific human genes were studied over the recent years in the Department of Molecular Bases of Human Genetics, Institute of Molecular Genetics. Clones Hfb1, Hmob3, and Hmob33 were selected from human brain cDNA libraries by differential screening. The clones were sequenced, mapped, and tested for expression in various human tissues. In vitro and in silico experiments identified Hfb1 as an earlier unknown complexin 2 gene (CPLX2) fragment, which codes for the large 3"-untranslated region of the CPLX2 mRNA. Hmob3 proved to correspond to an earlier unknown fragment of the large 3"-untranslated region of the human MAP1B mRNA. With Hfb1 and Hmob3, new terminal exons were revealed and exact structures established for CPLX2 and MAP1B. Hmob33 was identified as a fragment of the 3"-terminal exon of a new gene, MOB, which codes for a yet unknown evolutionarily conserved transmembrane protein. The structure of the deduced protein product was analyzed.     

镉胁迫下水稻OsPT1的表达及功能分析

OsPT1编码的水稻磷酸盐（Pi）转运蛋白在水稻生长发育、非生物胁迫应答等方面发挥重要的调控作用。前期研究表明OsPT1为镉（Cd）响应基因,但其在Cd胁迫下的功能及作用机制仍然未知。阐明OsPT1在Cd胁迫下的作用,并为低Cd水稻品种的选育奠定基础。通过生物信息学方法对该基因的序列特征、结构和功能进行分析和预测,利用实时荧光定量PCR（RT-qPCR）方法检测Cd胁迫下水稻不同组织、不同时间点OsPT1的相对表达量。此外,利用PCR的方法克隆OsPT1的编码序列,构建pGADT7-OsPT1重组质粒载体,并将其转入Δycf1 BY4741酵母菌株（Cd敏感酵母菌株）用以验证OsPT1对酵母Cd耐受性的影响。结果表明,OsPT1编码序列全长为1 584 bp,编码分子量为57.46 kD,由527个氨基酸构成的蛋白。在水稻基因组中该基因上游启动子区含有与光、厌氧、茉莉酸甲酯等环境和激素响应相关的调控元件。系统进化分析表明,水稻OsPT1与高粱SbPT1亲缘关系最近。基因的镉响应表达分析结果表明,与对照相比,经100 μmol/L Cd处理的水稻在1、6和12 h后,地上部分OsPT1的转录水平分别上调1.31、1.34和2.46倍;水稻根部OsPT1在处理1和6 h后分别上调1.28和1.14倍,但在Cd处理12 h后,其表达水平下调至处理前的0.62倍。转基因酵母Cd耐受性结果表明,与对照（0 μmol/L Cd）相比,经25 μmol/L Cd处理后,转OsPT1的酵母对Cd的耐受性有一定的下降。OsPT1可能在水稻应对Cd胁迫过程中发挥一定的作用。     

JunD的结构功能特征及其在肿瘤发展中的作用

JunD是一种属于多功能激活剂蛋白-1（activating protein-1,AP-1) 家族的转录因子,可以激活或抑制多种靶基因的表达.在生长发育过程中,在各种细胞类型中都呈现出组成性表达.近20年的临床数据及分子生物学研究表明,JunD蛋白的功能受多个复杂过程调控,包括转录控制、转录后调节、蛋白质翻译后修饰及蛋白-蛋白相互作用等.JunD基因表达的精细调控及JunD蛋白与其它蛋白之间的相互作用可调节细胞增殖、分化和凋亡等过程.JunD蛋白活性异常会导致肿瘤、代谢及病毒类疾病的发生.JunD蛋白的转录激活及抑制受1个复杂调控网络调控,在这个网络调节下,JunD蛋白在细胞的生长调控过程中发挥重要作用.本文就JunD基因表达的调控机制及其与肿瘤之间关系的最新研究进展做一综述.     

小麦TaLEA1基因的表达特征及抗逆功能验证

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁,因此耐盐基因挖掘对作物耐盐育种非常重要。许多研究表明胚胎发育晚期丰富蛋白(LEA)在植物应对非生物胁迫中发挥积极作用。本研究以小麦TaLEA1基因为研究对象,分析了其表达蛋白的理化性质及基因表达模式,并通过在拟南芥中过表达,分析Ta LEA1基因的抗逆功能。结果表明,TaLEA1基因的表达蛋白属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。Ta LEA1基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA1基因,显著提高了盐胁迫下转基因拟南芥的种子萌发率、根长及盐和旱胁迫下的叶绿素含量。本研究结果为LEA基因抗逆机理的研究和耐盐基因的挖掘提供了重要信息。