Polypyrimidine tract-binding protein 1 regulates the alternative splicing of dopamine receptor D(2)

Dopamine receptor D(2) (DRD2) has two splicing isoforms, a long form (D2L) and short form (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is unknown. In this study, we examined which splicing factors regulate the expression of D2L and D2S by over-expressing several RNA-binding proteins in HEK293 cells. In a cellular splicing assay, the over-expression of polypyrimidine tract-binding protein 1 (PTBP1) reduced the expression of D2S, whereas the knockdown of PTBP1 increased the expression of D2S. We also identified the regions of DRD2 that are responsive to PTBP1 using heterologous minigenes and deletion mutants. Our results indicate that PTBP1 regulates the alternative splicing of DRD2. Considering that DRD2 inhibits cAMP-dependent protein kinase A, which modulates the intracellular localization of PTBP1, PTBP1 may contribute to the autoregulation of DRD2 by regulating the expression of its isoforms.     

The CacyBP/SIP Protein is Sumoylated in Neuroblastoma NB2a Cells

The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.     

Arabidopsis thaliana proliferating cell nuclear antigen has several potential sumoylation sites

Proliferating cell nuclear antigen (PCNA) is post-translationally modified in yeast and animal cells. Major studies carried out in the last decade have focused on the role of sumoylated and ubiquitinated PCNA. Using different approaches, an interaction between plant PCNA and SUMO both in vivo and in bacteria has been demonstrated for the first time. In addition, identical sumoylation patterns for both AtPCNA1 and 2 were observed in bacteria. The plant PCNA sumoylation pattern has been shown to differ significantly from that of Saccharomyces cerevisiae. This result contrasts with a common opinion based on previous structural analysis of yeast, human, and plant PCNAs, which treats PCNA as a highly conserved protein even between species. Analyses of AtPCNA post-translational modifications using different SUMO proteins (SUMO1, 2, 3, and 5) revealed similar modification patterns for each tested SUMO protein. Potential target lysine residues that might be sumoylated in vivo were identified on the basis of in bacteria AtPCNA mutational analyses. Taken together, these results clearly show that plant PCNA is post-translationally modified in bacteria and may be sumoylated in a plant cell at various sites. These data open up important new perspectives for further detailed studies on the role of PCNA sumoylation in plant cells.     

多聚嘧啶区结合蛋白1通过调控基因的可变剪接促进胆管癌细胞的生长、迁移及侵袭能力

胆管癌是一种起病隐匿、侵袭性强、致死率高的原发性恶性肿瘤。多聚嘧啶区结合蛋白1(polypyrimidine tract-binding protein 1, PTBP1)已被报道,在多种类型肿瘤组织中异常高表达并参与癌症进展,但其在胆管癌中的作用仍未见报道。该研究旨在探讨PTBP1在胆管癌中的生物学功能,并初步解析其分子机制。本文利用公开的癌症基因组图谱(the cancer genome atlas, TCGA)数据,分析了胆管癌及癌旁组织中的PTBP1 mRNA表达水平。结果显示,PTBP1在胆管癌组织中的表达水平显著高于癌旁组织(P < 0.05)。随后,在胆管癌细胞系RBE和HuH28中,通过CCK-8和细胞平板克隆实验,评价了PTBP1对胆管癌细胞生长能力的影响。结果显示,过表达PTBP1可显著促进胆管癌细胞的生长(P < 0.01),而敲低PTBP1显著抑制胆管癌细胞的生长(P < 0.001)。Transwell和Invasion实验结果显示,过表达PTBP1可显著促进胆管癌细胞的迁移和侵袭(P < 0.001),而敲低PTBP1显著抑制胆管癌细胞的迁移和侵袭(P < 0.001)。转录物组测序和通路富集分析结果显示,在胆管癌细胞中,敲低PTBP1后上调表达的基因显著富集于p53信号通路;而下调表达的基因显著富集于胆固醇代谢、Rho GTPase和TGF-β等信号通路。基于上述转录物组测序数据,本文还分析发现,敲低PTBP1可导致一系列基因发生异常的mRNA可变剪接事件,例如参与TGF-β调控的TGIF1及与p53活性相关的GNAS基因等。综上所述,PTBP1可能通过调控一系列基因的可变剪接而影响多个癌症相关的信号通路,从而促进胆管癌的进展。     

The effects of PTBP3 silencing on the proliferation and differentiation of MKN45 human gastric cancer cells

Aims

PTBP3 overexpression inhibits the differentiation of leukemia cells; however, its effects on the differentiation and proliferation of solid cancer cells remain unclear. Thus, the impact of PTBP3 on the differentiation and proliferation of gastric cancer cells was investigated.

Main methods

PTBP3 expression was analyzed in normal and tumor tissues using immunohistochemistry. A xenograft model was established in nude mice by subcutaneous injection of untransfected human gastric cancer MKN45 cells or those expressing a control vector or PTBP3 siRNA. We analyzed the tumor inhibition rate, the expression of PTBP3, the PCNA-positive rate and the serum levels of CEA, CA199, CA125, LDH, ALP and γ-GT in different groups.

Key findings

The tumor weights in the PTBP3 siRNA group were significantly lower than that of the MKN45 cell control group (P < 0.001). Immunohistochemistry analysis of PCNA expression revealed that it was markedly reduced after PTBP3 silencing. ELISAs showed that the serum levels of CEA and CA199 tumor markers as well as LDH and ALP were reduced after PTBP3 silencing. Transmission electron microscopy revealed that MKN45 cells expressing PTBP3 siRNA had reduced nuclear-to-cytoplasmic ratio and regular nuclei, suggesting differentiation.

Significance

PTBP3 may promote proliferation and inhibit the differentiation of human gastric cancer MKN45 cells.     

The Ulp1 SUMO isopeptidase: distinct domains required for viability,nuclear envelope localization,and substrate specificity

Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A approximately 200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.     

PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy

Invasion and migration is the hallmark of malignant tumors as well as the major cause for breast cancer death. The polypyrimidine tract binding, PTB, protein serves as an important model for understanding how RNA binding proteins affect proliferation and invasion and how changes in the expression of these proteins can control complex programs of tumorigenesis. We have investigated some roles of polypyrimidine tract binding protein 1 (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in breast cancer tissues compared with normal tissues and the same result was confirmed in breast cancer cell lines. Knockdown of PTBP1 substantially inhibited tumor cell growth, migration, and invasion. These results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of metastasis. We further analyzed the relationship between PTBP1 and clinicopathological parameters and found that PTBP1 was correlated with her‐2 expression, lymph node metastasis, and pathological stage. This will be a novel target for her‐2(⁺) breast cancer. PTBP1 exerts these effects, in part, by regulating the phosphatase and tensin homolog‐phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/protein kinase B (PTEN‐PI3K/Akt) pathway and autophagy, and consequently alters cell growth and contributes to the invasion and metastasis.     

Retracted: Long noncoding RNA HCG22 suppresses proliferation and metastasis of bladder cancer cells by regulation of PTBP1

Multifarious biological functions of long noncoding RNAs (lncRNAs) have been reported in various cancers including bladder cancer (BCa). This study aims to determine the biological role of a certain lncRNA in BCa. Consistent with the data of The Cancer Genome Atlas database, it was validated that lncRNA HLA complex group 22 (HCG22) was weakly expressed in BCa samples and lowly expressed HCG22 was closely correlated with low overall survival of the BCa patient. To verify the role of HCG22 in BCa progression, functional experiments were carried out in two representative BCa cells (J82 and T24) and the negative effects of HCG22 expression on the cell proliferation, migration, and epithelial–mesenchymal transition were identified. Mechanistically, polypyrimidine tract-binding protein 1 (PTBP1), which was highly expressed in BCa tissues and cell lines, was negatively regulated by HCG22 and the PTBP1-mediated Warburg effect was also obstructed by HCG22. Furthermore, HCG22 modulated the expression of PTBP1 through destabilizing human antigen R (HuR). And functional rescue assays confirmed that HCG22 functioned in bladder cancer through downregulating PTBP1. In conclusion, the present study revealed that HCG22 inhibited BCa progression via the HuR/PTBP1 axis, opening new prospects for potent therapeutic regimens for BCa patients.     

SUMO-targeted ubiquitin ligases

Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or STUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

The S. pombe Translation Initiation Factor eIF4G Is Sumoylated and Associates with the SUMO Protease Ulp2

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively.     

Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins

SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.     

Sumoylation inhibits alpha-synuclein aggregation and toxicity

Posttranslational modification of proteins by attachment of small ubiquitin-related modifier (SUMO) contributes to numerous cellular phenomena. Sumoylation sometimes creates and abolishes binding interfaces, but increasing evidence points to another role for sumoylation in promoting the solubility of aggregation-prone proteins. Using purified α-synuclein, an aggregation-prone protein implicated in Parkinson's disease that was previously reported to be sumoylated upon overexpression, we compared the aggregation kinetics of unmodified and modified α-synuclein. Whereas unmodified α-synuclein formed fibrils, modified α-synuclein remained soluble. The presence of as little as 10% sumoylated α-synuclein was sufficient to delay aggregation significantly in vitro. We mapped SUMO acceptor sites in α-synuclein and showed that simultaneous mutation of lysines 96 and 102 to arginine significantly impaired α-synuclein sumoylation in vitro and in cells. Importantly, this double mutant showed increased propensity for aggregation and cytotoxicity in a cell-based assay and increased cytotoxicity in dopaminergic neurons of the substantia nigra in vivo. These findings strongly support the model that sumoylation promotes protein solubility and suggest that defects in sumoylation may contribute to aggregation-induced diseases.     

Ubiquitin-dependent proteolytic control of SUMO conjugates

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.