视黄醇结合蛋白研究

人视黄醇结合蛋白 (ｒｅｔｉｎｏｌｂｉｎｄｉｎｇｐｒｏ ｔｅｉｎ ,ＲＢＰ)由 1 84个氨基酸组成 ,分子量 2 1ｋｄ ,是单链、疏水小分子结合蛋白 ,属于脂肪酸结合蛋白家族成员。ＲＢＰ在肝脏中合成 ,释放入血后与视黄醇 (ＲＯＨ)、甲状腺素运载蛋白(ＴＴＲ)以 1∶1∶1的比例形成三元复合物 ,是体内运送视黄醇至其特定靶组织的运载蛋白[1] 。ＲＢＰ的ｍＲＮＡ在肝中的含量最高。ＲＢＰ只有受到视黄醇的刺激才分泌 ,视黄醇的缺乏会阻断细胞内合成的ＲＢＰ由内质网到高尔基体的转运 ,从而影响ＲＢＰ的分泌 ,而且分泌入血的ＲＢＰ量也受到视黄醇…     

视黄醇结合蛋白的结构与功能

视黄醇结合蛋白（RBP）是视黄醇转运的载体蛋白,作为结合小分子流水物质的载体蛋白家族（lipocalin）的一个重要成员,其结构与功能的研究正受到国外学者的重视,文章介绍了视黄醇结合蛋白的性质、结构研究进展,讨论了视黄醇结合蛋白与前蛋白和受体相互作用的位点和结构特点．     

视黄醇结合蛋白4在病理妊娠中的研究进展

视黄醇结合蛋白4(Retinol binding protein 4,RBP4)是近年来新发现的一种脂肪细胞因子,在动物与人类的研究中均发现其与胰岛素抵抗的发生及糖、脂代谢的调节密切相关,随着机体胰岛素抵抗及糖脂代谢异常程度的增加,RBP4水平上升。在妊娠期,母体脂肪量增加并伴有不同程度的胰岛素抵抗。在正常妊娠时,RBP4随着妊娠的进展而升高,且与胰岛素抵抗程度增加相一致。与正常妊娠相比,妊娠期糖尿病(gestational diabetes mellitus,GDM)患者的RBP4升高更明显,且RBP4的升高水平与GDM预后相关。研究发现早期RBP4的升高可以预测GDM的发生,此外控制RBP4水平可能干预GDM发展。RBP4在妊娠期高血压疾病中升高;RBP4在脐血中的水平与胎儿生长密切相关,在小于胎龄儿中水平下降,在大于胎龄儿中升高;RBP4与早产的关系并不明确。进一步研究RBP4在病理妊娠的作用机制,可为病理妊娠的发生发展和防治提供新方向。     

Liver Retinol Transporter and Receptor for Serum Retinol-binding Protein (RBP4)

Vitamin A (retinol) is absorbed in the small intestine, stored in liver, and secreted into circulation bound to serum retinol-binding protein (RBP4). Circulating retinol may be taken up by extrahepatic tissues or recycled back to liver multiple times before it is finally metabolized or degraded. Liver exhibits high affinity binding sites for RBP4, but specific receptors have not been identified. The only known high affinity receptor for RBP4, Stra6, is not expressed in the liver. Here we report discovery of RBP4 receptor-2 (RBPR2), a novel retinol transporter expressed primarily in liver and intestine and induced in adipose tissue of obese mice. RBPR2 is structurally related to Stra6 and highly conserved in vertebrates, including humans. Expression of RBPR2 in cultured cells confers high affinity RBP4 binding and retinol transport, and RBPR2 knockdown reduces RBP4 binding/retinol transport. RBPR2 expression is suppressed by retinol and retinoic acid and correlates inversely with liver retinol stores in vivo. We conclude that RBPR2 is a novel retinol transporter that potentially regulates retinol homeostasis in liver and other tissues. In addition, expression of RBPR2 in liver and fat suggests a possible role in mediating established metabolic actions of RBP4 in those tissues.     

Genetic Variation in ANGPTL4 Provides Insights into Protein Processing and Function

Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that modulates the disposition of circulating triglycerides (TG) by inhibiting lipoprotein lipase (LPL). Here we examine the steps involved in the synthesis and post-translational processing of ANGPTL4, and the effects of a naturally occurring sequence variant (E40K) that is associated with lower plasma TG levels in humans. Expression of the wild-type and mutant proteins in HEK-293A cells indicated that ANGPTL4 formed dimers and tetramers in cells prior to secretion and cleavage of the protein. After cleavage at a canonical proprotein convertase cleavage site (¹⁶¹RRKR¹⁶⁴), the oligomeric structure of the N-terminal domain was retained whereas the C-terminal fibrinogen-like domain dissociated into monomers. Inhibition of cleavage did not interfere with oligomerization of ANGPTL4 or with its ability to inhibit LPL, whereas mutations that prevented oligomerization severely compromised the capacity of the protein to inhibit LPL. ANGPTL4 containing the E40K substitution was synthesized and processed normally, but no monomers or oligomers of the N-terminal fragments accumulated in the medium; medium from these cells failed to inhibit LPL activity. Parallel experiments performed in mice recapitulated these results. Our findings indicate that oligomerization, but not cleavage, of ANGPTL4 is required for LPL inhibition, and that the E40K substitution destabilizes the protein after secretion, preventing the extracellular accumulation of oligomers and abolishing the ability of the protein to inhibit LPL activity.Angiopoietin-like protein 4 (ANGPTL4)⁴ is a 50-kDa protein that is synthesized and secreted from several metabolically active tissues and has been implicated in the trafficking of circulating TG (1, 2). Triglycerides, either acquired from the diet or synthesized endogenously, circulate in blood as constituents of chylomicrons and very low density lipoproteins (VLDL). As these lipoproteins circulate in tissues they encounter lipoprotein lipase (LPL) at the vascular endothelial surfaces. LPL hydrolyzes the TG, producing free fatty acids that are taken up by the surrounding tissues. ANGPTL4 inhibits the activity of LPL, thereby limiting the uptake of TG-derived fatty acids by the underlying cells (3, 4). Overexpression of ANGPTL4 in mice causes severe hypertriglyceridemia, whereas mice lacking ANGPTL4 have increased LPL activity and low plasma levels of TG (5, 6). In mice, ANGPTL4 is predominantly expressed in adipose tissue and is strongly induced by fasting (2). Accordingly it has been proposed that ANGPTL4 inhibits LPL activity in adipose tissue to reroute fatty acids away from fat to muscle and other tissues when food intake is low (3, 4).ANGPTL4 belongs to a family of seven structurally similar secreted proteins (ANGPTL1-ANGPTL7) that contain a signal sequence followed by an α-helical region predicted to form a coiled-coil, and a globular fibrinogen-like domain at the C terminus (1). Gel filtration studies of recombinant ANGPTL4 indicate that the protein assembles into oligomers that are stabilized by disulfide bonds (7). Substitution of two highly conserved cysteine residues at positions 76 and 80 in the α-helical domain prevents oligomerization of ANGPTL4 and impairs the ability of the recombinant protein to increase plasma TG levels when overexpressed in the livers of rats (7).Upon secretion into the circulation, ANGPTL4 is cleaved into an N-terminal domain and a C-terminal fibrinogen-like domain (8). The N-terminal peptide circulates as an oligomer, and the fibrinogen-like domain circulates as a monomer (8). The N-terminal helical region of ANGPTL4 is necessary and sufficient for inhibition of LPL (9). A peptide corresponding to amino acids 1-187 of the protein binds LPL with high affinity and converts the enzyme from catalytically active dimers to inactive monomers, thereby inhibiting LPL activity (10). After disrupting the LPL dimer, ANGPTL4 is released. The LPL monomers remain folded and stable but fail to re-form active dimers. These data suggest that the N-terminal domain of ANGPTL4 interacts directly but transiently with LPL, triggering a stable conformational switch in LPL that irreversibly inactivates the enzyme.Recently, we used a population-based resequencing strategy to examine the metabolic role of ANGPTL4 in humans (11). Resequencing the coding region of ANGPTL4 in a large (n = 3,501), multiethnic sample revealed multiple rare sequence variations that alter an amino acid in the protein and are associated with low plasma TG levels. In addition, we identified a more common variant (E40K), that was present in ∼3% of European-Americans and was associated with significantly lower plasma levels of TG and low density lipoprotein-cholesterol (LDL-C), and higher levels of high density lipoprotein (HDL)-C in two large epidemiological studies (11). These association studies confirmed that ANGPTL4 is involved in TG metabolism in humans, and also revealed additional roles in humans in the metabolism of HDL and LDL, which were not apparent from studies in genetically modified mice.Here we examined the synthesis, secretion, and processing of ANGPTL4 and determine the mechanism by which substitution of a basic (lysine) for an acidic (glutamate) residue at residue 40 affects the function of the protein.     

Using Superfolder Green Fluorescent Protein for Periplasmic Protein Localization Studies

Studies investigating the subcellular localization of periplasmic proteins have been hampered by problems with the export of green fluorescent protein (GFP). Here we show that a superfolding variant of GFP (sfGFP) is fluorescent following Sec-mediated transport and works best when the cotranslational branch of the pathway is employed.     

Arabidopsis DELLA Protein Degradation Is Controlled by a Type-One Protein Phosphatase,TOPP4

Gibberellins (GAs) are a class of important phytohormones regulating a variety of physiological processes during normal plant growth and development. One of the major events during GA-mediated growth is the degradation of DELLA proteins, key negative regulators of GA signaling pathway. The stability of DELLA proteins is thought to be controlled by protein phosphorylation and dephosphorylation. Up to date, no phosphatase involved in this process has been identified. We have identified a dwarfed dominant-negative Arabidopsis mutant, named topp4-1. Reduced expression of TOPP4 using an artificial microRNA strategy also resulted in a dwarfed phenotype. Genetic and biochemical analyses indicated that TOPP4 regulates GA signal transduction mainly via promoting DELLA protein degradation. The severely dwarfed topp4-1 phenotypes were partially rescued by the DELLA deficient mutants rga-t2 and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for the biological function of TOPP4. Both RGA and GAI were greatly accumulated in topp4-1 but significantly decreased in 35S-TOPP4 transgenic plants compared to wild-type plants. Further analyses demonstrated that TOPP4 is able to directly bind and dephosphorylate RGA and GAI, confirming that the TOPP4-controlled phosphorylation status of DELLAs is associated with their stability. These studies provide direct evidence for a crucial role of protein dephosphorylation mediated by TOPP4 in the GA signaling pathway.     

Protein Phosphorylation during Coconut Zygotic Embryo Development

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-³²P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [³²P]phosphoserine, [³²P]phosphothreonine, and [³²P]phosphotyrosine in [³²P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60^src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species.     

A Role for Protein Phosphorylation in Cytochrome P450 3A4 Ubiquitin-dependent Proteasomal Degradation

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr²⁶⁴ and Ser⁴²⁰. We now document that liver cytosolic kinases additionally target Ser⁴⁷⁸ as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Δ strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser⁴⁷⁸, Thr²⁶⁴, and Ser⁴²⁰ residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.Hepatic cytochromes P450 (P450s)³ are integral endoplasmic reticulum (ER)-anchored hemoproteins engaged in the oxidative biotransformation of various endo- and xenobiotics. Of these, human CYP3A4 is the most dominant liver enzyme, accounting for >30% of the hepatic microsomal P450 complement, and responsible for the oxidative metabolism of over 50% of clinically relevant drugs (1). In common with all the other ER-bound P450s, CYP3A4 is a monotopic protein with its N-terminal ≈33-residue domain embedded in the ER membrane with the bulk of its structure in the cytosol. Our in vivo studies of the heterologously expressed CYP3A4 in the yeast Saccharomyces cerevisiae as well as of its rat liver CYP3A2/3A23 orthologs in primary hepatocytes have revealed that human and rat liver CYPs 3A are turned over via ubiquitin (Ub)-dependent proteasomal degradation (UPD) (2–8). Thus, CYPs 3A represent excellent prototypic substrates of ER-associated degradation (ERAD), specifically of the ERAD-C pathway (6–11). Consistent with this CYP3A ERAD process, our studies of in vivo and/or in vitro reconstituted systems have led us to conclude that CYPs 3A are ubiquitinated by the UBC7/gp78 Ub-ligase complex and recruited by the p97-Npl4-Ufd1 complex before their degradation by the 26 S proteasome (4–8, 12). Because all these processes are energy-dependent, it is not surprising that in vitro reconstitution of CYP3A4 UPD requires ATP. However, inclusion of γ-S-[³²P]ATP in an in vitro reconstituted CYP3A4 ubiquitination system catalyzed by rat liver cytosolic fraction II (FII) resulted in CYP3A4 protein phosphorylation, i.e. γ-[³²P]phosphoryl transfer onto CYP3A4 target residues (13, 14). This phosphorylation was enhanced after cumene hydroperoxide (CuOOH)-mediated CYP3A4 inactivation. The physiological role, if any, of this CYP3A4 post-translational modification is unclear.CYP3A4 is not the only P450 that is phosphorylated. Since the in vitro phosphorylation of a hepatic P450 (CYP2B4) by cAMP-dependent protein kinase A (PKA) was first described (15), various P450s, particularly those belonging to the subfamily 2, were documented to be phosphorylated in cell-free systems, hepatocyte incubations, and intact animals (16–32). Common features of such P450 phosphorylation were the presence of a cytosolically exposed PKA recognition sequence (RRXS) with the Ser residue as the exclusive kinase target, and the ensuing loss of prosthetic heme, conversion to the inactive P420 species, and consequent dramatic functional inactivation (15–20). Studies in intact rats also identified CYPs 3A and 2C6 as kinase targets (21). Although both these P450s lack the hallmark PKA recognition sequence, apparently they possess secondary PKA targeting sequences or are phosphorylated by other protein kinases such as PKC. Indeed, in vitro studies revealed that P450s were phosphorylated in an isoform-dependent manner by either PKA or PKC, except for CYP2B1, which was heavily phosphorylated by both (20). Over the years since this particular post-translational P450 modification was recognized, it has been assigned various functional roles (17, 29–33). Among these, as first proposed by Taniguchi et al. (16) and later explored both by Eliasson et al. (23–26) and us (13, 14), P450 phosphorylation served as a marker for its degradation. Accordingly, the phosphorylation of CYP2E1Ser¹²⁹ and CYP3A1Ser³⁹³ by a microsomal cAMP-dependent protein kinase has been proposed to predispose these P450s but not the similarly phosphorylated CYP2B1 to proteolytic degradation by an integral ER Mg²⁺-ATP-activated serine protease (23–27). However, heterologous expression of CYP2E1S129A/S129G site-directed mutants in COS7 cells apparently had no effect on its relative stability thereby revealing that if CYP2E1 phosphorylation is important for its degradation (34, 35), then alternate Ser/Thr residues (i.e. in plausible secondary PKA recognition sites, Lys-Lys-Ser²⁰⁹-Lys and Lys-Lys-Ser⁴⁴⁹-Ala) may be recruited.On the other hand, on the basis of rapid phosphorylation of CuOOH-inactivated CYP3A4 that precedes its ubiquitination and 26 S proteasomal degradation in an in vitro liver cytosolic FII-catalyzed system, we have proposed that CYP3A4 phosphorylation was essential for targeting it to proteins participating in its UPD/ERAD (13). Indeed, several examples of similar phosphorylation for targeting proteins to UPD exist, of which IκBα phosphorylation is the most notable and perhaps the best documented (36–47; see “Discussion”).Our in vitro studies with specific kinase inhibitors as probes identified both PKC and PKA as the major FII kinases responsible for CYP3A4 phosphorylation (14). Indeed, in vitro model studies of CYP3A4 with PKC as the kinase, coupled with lysylendopeptidase C (Lys-C) digestion of the phosphorylated protein and liquid chromatography-tandem mass spectrometric (LC-MS/MS) analyses of the Lys-C digests, identified two PKC-phosphorylated CYP3A4 peptides ²⁵⁸ESRLEDpTQK²⁶⁶ and ⁴¹⁴FLPERFpSK⁴²¹ unambiguously phosphorylated at Thr²⁶⁴ and Ser⁴²⁰ (14). These same residues were also phosphorylated in corresponding studies with PKA.⁴ Furthermore, although both native and CuOOH-inactivated CYP3A4 were phosphorylated at Thr²⁶⁴, Ser⁴²⁰ phosphorylation was particularly enhanced after CuOOH-mediated CYP3A4 inactivation (14). Corresponding studies of CuOOH-inactivated CYP3A4 using rat liver cytosolic FII as the source of the kinase(s), revealed ³²P phosphorylation of both these peptides as well as that of an additional CYP3A4 peptide ⁴⁷⁷LS(p)LGGLLQPEKPVVLK⁴⁹². Unlike the unambiguous mass spectrometric identification of Thr²⁶⁴ and Ser⁴²⁰ as the phosphorylated CYP3A4 residues, the phosphorylation of Ser⁴⁷⁸, the only plausible phosphorylatable residue in this ³²P-labeled peptide, was not similarly established. Nevertheless, the predominant phosphorylation of Thr²⁶⁴ in native CYP3A4 (14), but of two additional residues in the CuOOH-inactivated enzyme, is consistent with the inactivation-induced structural unraveling of this enzyme with exposure of otherwise concealed and/or kinase-inaccessible domains (48). Such unraveling of CYP3A4 protein stems from the irreversible modification of its active site by fragments generated from CuOOH-mediated oxidative destruction of its prosthetic heme (49). In this study, using mass spectrometric analyses of Lys-C digests of FII-phosphorylated CYP3A4, we have provided unambiguous evidence that in addition to Thr²⁶⁴ and Ser⁴²⁰, Ser⁴⁷⁸ is indeed phosphorylated. More importantly, through alanine-scanning mutagenesis of these three residues, we now document that although neither the structural conformation nor the catalytic function of this triple CYP3A4T264A/S420A/S478A mutant is altered, its degradation after heterologous expression in S. cerevisiae is significantly impaired. This is also true of CYP3A4T264A/S420A/S478A mutant degradation in human embryonic kidney (HEK293T) cells. Furthermore, using an in vitro reconstituted CYP3A4 ubiquitination system, catalyzed by human Ub-conjugating E2 enzyme UBC7 and integral ER protein gp78 as the E3 Ub ligase (12), we document that PKA/PKC-mediated phosphorylation of the wild type CYP3A4 (CYP3A4wt) considerably enhanced its UBC7/gp78-mediated ubiquitination. Together these findings reveal the critical importance of CYP3A4 phosphorylation at these residues for its UPD and suggest a direct link between phosphorylation and its ubiquitination and degradation.     

随机尿、晨尿视黄醇结合蛋白对早期肾功能损伤的诊断价值

目的:探究随机尿、晨尿视黄醇结合蛋白对早期肾功能损伤的诊断价值.方法:选取我院2011年8月-2012年9月收治的48例Ⅱ型糖尿病患者,按照随机数字表进行平均分组,随机尿组24例,选取任意时段尿液标本,晨尿组24例,选取清晨时段尿液标本.对两组患者尿液标本进行尿视黄醇结合蛋白(Urinary Retinol-binding Protein,U-RBP)、尿微量白蛋白(Urinary Microalbum, U-mAlb)及尿N-乙酰-β-D氨基葡萄糖苷酶(Urinary N-acetyl beta-D-Glucosaminidase,NAG)水平测定,进行相关性分析并观察两组诊断阳性率.结果:随机尿组U-RBP、U-mAlb及NAG分别为(1.7± 0.9) mg/L、(76.2± 41.5) mg/L及(41.2± 30.0) U/L;晨尿组U-RBP、U-mAlb及NAG分别为(3.6±1.2)mgL、(118.5±71.)mg/L及(116.5±71.9) U/L.两组患者尿液检测指标均高于正常值,且晨尿组指标较随机尿组更高,两组数据对比存在统计学差异;随机尿组U-RBP、U-mAlb及NAG阳性例数分别为12例、6例及4例,晨尿组U-RBP、U-mAlb及NAG阳性例数分别为17例、13例及11例,晨尿组U-RBP、U-mAlb及NAG阳性率均高于随机尿组,两组数据对比存在统计学差异;随机尿组U-RBP与肾损伤相关性r=0.532,P ＞0.05,无明显相关性;晨尿组U-RBP与肾损伤相关性r=0.867,P＜0.01,呈高度正相关.结论:随机尿及晨尿均可指示患者肾损伤出现,但随机尿蛋白指标无法有效指示肾损伤程度,对早期肾功能损害确诊准确率有限,而晨尿尿蛋白正常排泄率更高,且能够有效指示肾损伤程度,适用于糖尿病早期肾功能损伤的诊断及监测.     

Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.The excision of intronic sequences from precursor mRNAs is a critical step during eukaryotic gene expression. This reaction is catalyzed by the spliceosome, a macromolecular complex composed of small nuclear ribonucleoproteins (snRNPs) and many additional proteins. Spliceosome assembly and splicing catalysis occur in an ordered multistep process, which includes multiple conformational rearrangements (35). Spliceosomal snRNPs are assembled from snRNAs and protein components, the latter of which fall into two classes: snRNP-specific and common proteins. The common or canonical core proteins are also termed Sm proteins, specifically SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG (10; reviewed in reference 9), which all share an evolutionarily conserved bipartite sequence motif (Sm1 and Sm2) required for Sm protein interactions and the formation of the heteroheptameric Sm core complex around the Sm sites of the snRNAs (3, 7, 29). Prior to this, the Sm proteins form three heteromeric subcomplexes: SmD3/SmB, SmD1/SmD2, and SmE/SmF/SmG (23; reviewed in reference 34). Individual Sm proteins or Sm subcomplexes cannot stably interact with the snRNA. Instead, a stable subcore forms by an association of the subcomplexes SmD1/SmD2 and SmE/SmF/SmG with the Sm site on the snRNA; the subsequent integration of the SmD3/SmB heterodimer completes Sm core assembly.In addition to the canonical Sm proteins, other proteins carrying the Sm motif have been identified for many eukaryotes. Those proteins, termed LSm (like Sm) proteins, exist in distinct heptameric complexes that differ in function and localization. For example, a complex composed of LSm1 to LSm7 (LSm1-7) accumulates in cytoplasmic foci and participates in mRNA turnover (4, 8, 31). Another complex, LSm2-8, binds to the 3′ oligo(U) tract of the U6 snRNA in the nucleus (1, 15, 24). Finally, in the U7 snRNP, which is involved in histone mRNA 3′-end processing, the Sm proteins SmD1 and SmD2 are replaced by U7-specific LSm10 and LSm11 proteins, respectively (20, 21; reviewed in reference 28).This knowledge is based primarily on the mammalian system, where spliceosomal snRNPs are biochemically well characterized (34). In contrast, for trypanosomes, comparatively little is known about the components of the splicing machinery and their assembly and biogenesis. In trypanosomes, the expression of all protein-encoding genes, which are arranged in long polycistronic units, requires trans splicing. Only a small number of genes are additionally processed by cis splicing (reviewed in reference 11). During trans splicing, a short noncoding miniexon, derived from the spliced leader (SL) RNA, is added to each protein-encoding exon. Regarding the trypanosomal splicing machinery, the U2, U4/U6, and U5 snRNPs are considered to be general splicing factors, whereas the U1 and SL snRNPs represent cis- and trans-splicing-specific components, respectively. In addition to the snRNAs, many protein splicing factors in trypanosomes have been identified based on sequence homology (for example, see references 14 and 19).Recent studies revealed variations in the Sm core compositions of spliceosomal snRNPs from Trypanosoma brucei. Specifically, in the U2 snRNP, two of the canonical Sm proteins, SmD3 and SmB, are replaced by two novel, U2 snRNP-specific proteins, Sm16.5K and Sm15K (33). In this case, an unusual purine nucleotide, interrupting the central uridine stretch of the U2 snRNA Sm site, discriminates between the U2-specific and the canonical Sm cores. A second case of Sm core variation was reported for the U4 snRNP, in which a single protein, SmD3, was suggested to be replaced by the U4-specific LSm protein initially called LSm2, and later called SSm4, based on a U4-specific destabilization after SSm4 knockdown (30). A U4-specific Sm core variation was also previously suggested and discussed by Wang et al. (33), based on the inefficient pulldown of U4 snRNA through tagged SmD3 protein. However, neither of these two studies conclusively demonstrated by biochemical criteria that the specific Sm protein resides in the U4 Sm core; a copurification of other snRNPs could not be unequivocally ruled out.By using a combination of RNase H cleavage, tandem affinity purification, and mass spectrometry, we provide here direct biochemical evidence that in the variant Sm core of the U4 snRNP, only SmD3 is replaced by the U4-specific SSm4. SSm4 is nuclear localized, and the silencing of SSm4 leads to a characteristic phenotype: dramatic growth inhibition, general trans- and cis-splicing defects, a loss of the integrity of the U4 snRNA, as well as a destabilization of the U4/U6 di-snRNP. Furthermore, in vitro reconstitution assays revealed that under stringent conditions, SSm4 is sufficient to specify U4-specific Sm core assembly. In sum, our data establish SSm4 as a specific component of the U4 Sm core and demonstrate its importance in U4/U6 di-snRNP biogenesis, splicing function, and cell viability.     

Mismatch Repair Protein Deficiency Compromises Cisplatin-induced Apoptotic Signaling

Mismatch repair (MMR) proteins participate in cytotoxicity induced by certain DNA damage-inducing agents, including cisplatin (cis-diamminedichloroplatinum(II), CDDP), a cancer chemotherapeutic drug utilized clinically to treat a variety of malignancies. MMR proteins have been demonstrated to bind to CDDP-DNA adducts and initiate MMR protein-dependent cell death in cells treated with CDDP; however, the molecular events underlying this death remain unclear. As MMR proteins have been suggested to be important in clinical responses to CDDP, a clear understanding of MMR protein-dependent, CDDP-induced cell death is critical. In this report, we demonstrate MMR protein-dependent relocalization of cytochrome c to the cytoplasm and cleavage of caspase-9, caspase-3, and poly(ADP-ribose) polymerase upon treatment of cells with CDDP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent cytotoxicity, suggesting that a caspase-dependent signaling mechanism is required for the execution of this cell death. p53 protein levels were up-regulated independently of MMR protein status, suggesting that p53 is not a mediator of MMR-dependent, CDDP-induced death. This work is the first indication of a required signaling mechanism in CDDP-induced, MMR protein-dependent cytotoxicity, which can be uncoupled from other CDDP response pathways, and defines a critical contribution of MMR proteins to the control of cell death.The MMR² system of proteins plays roles in diverse cellular processes, perhaps most notably in preserving genomic integrity by recognizing and facilitating the repair of post-DNA replication base pairing errors. Recognition of these errors and recruitment of repair machinery is performed by the MutSα complex (consisting of the MMR proteins MSH2 and MSH6) or MutSβ complex (consisting of MSH2 and MSH3). Defects in MMR proteins render cells hypermutable and promote microsatellite instability, a hallmark of MMR defects. MMR protein defects are found in a wide variety of sporadic cancers, as well as in hereditary non-polyposis colorectal cancer (1).In addition to their role in DNA repair, MMR proteins also play a role in cytotoxicity induced by specific types of DNA-damaging chemotherapeutic drugs, such as CDDP, which is utilized clinically to treat a number of different cancer types. MutSα recognizes multiple types of DNA damage, including 1,2-intrastrand CDDP adducts and O⁶-methylguanine lesions (2). Treatment of cells with compounds that induce these types of lesions, including CDDP and methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), results in MMR protein-dependent cell cycle arrest and cell death (3–7). This suggests that MMR proteins, in addition to their role in DNA repair, are also capable of initiating cell death in response to certain types of DNA damage.Cells treated with DNA-damaging agents frequently activate an apoptotic cell death pathway mediated by the mitochondria. This intrinsic death signaling pathway predominantly involves the coordinated activity of two groups of proteins: pro-death members of the Bcl-2 family that control the integrity of mitochondrial membranes, and members of the caspase family of cysteinyl proteases that proteolytically cleave intracellular substrates, giving rise to apoptotic morphology and destruction of the cell (8, 9). Pro-death Bcl-2 family members, such as Bax and Bak, target the outer mitochondrial membrane and cause the cytosolic release of pro-death factors residing within the mitochondria of unstressed cells (8). Predominant among these factors is cytochrome c, whose cytoplasmic localization results in the formation of a caspase-activating platform known as the apoptosome (10). This complex includes the adaptor protein Apaf-1, and when formed the apoptosome promotes the cleavage and activation of caspase-9 (11, 12). Once activated, this apical caspase proceeds to cleave and activate caspase-3, the predominant effector protease of apoptosis.A significant amount of evidence has been gathered illustrating MMR protein-dependent pro-death signaling in response to methylating agents (13–16, 3). In contrast, the MMR protein-dependent cytotoxic response to CDDP is largely unknown, with only the p53-related transactivator protein p73 and the c-Abl kinase clearly implicated as potential mediators of CDDP/MMR protein-dependent cell death in human cells (17, 18). Interestingly, ATM, Chk1, Chk2, and p53, which are activated in an MMR protein-dependent manner after treatment of cells with MNNG (3, 13), are not involved in the MMR-dependent response to CDDP (7, 17). In addition, the magnitude of MMR protein-dependent cell death induced by methylating agents and CDDP differs (4). These findings suggest that unique signaling pathways may be engaged by MMR proteins depending upon the type of recognized lesion. As such, there is a requirement for further study of the molecular events underlying MMR protein-dependent cell death and cell cycle arrest for each type of recognized DNA lesion. This is particularly relevant in the case of CDDP, as evidence from a limited number of retrospective clinical studies suggests that MMR proteins play an important role in patient response to CDDP. Several studies examining immunohistochemical staining against MSH2 or MLH1 have demonstrated that levels of these proteins are reduced in ovarian and esophageal tumor samples following CDDP-based chemotherapy (19, 20). Low levels of MMR protein post-chemotherapy seem to be predictive of lower overall survival in a certain subset of tumors (esophageal cancer), but not others (ovarian and non-small cell lung cancer) (19–21). Two recent studies examining MMR protein levels and microsatellite instability in germ cell tumors from patients receiving platinum-based chemotherapy have suggested a prognostic value for pre-chemotherapy MMR protein status in these tumors (22, 23). This potential clinical relevance underscores the need for a greater understanding of MMR protein-dependent mechanisms of CDDP-induced cell death.In this study, we report that CDDP induces an MMR protein-dependent decrease in cell viability and MMR protein-dependent signaling in the form of cytochrome c release to the cytoplasm and cleavage of caspase-9, caspase-3, and PARP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent loss of cell viability, indicating a requirement for caspase activation in this process and uncoupling MMR protein-dependent cytotoxic signaling from other CDDP response pathways. Additionally, the CDDP-induced, MMR protein-dependent cytotoxic response is independent of p53 signaling. Our results demonstrate for the first time an MMR protein-dependent pro-death signaling pathway in cells treated with CDDP.     

A Proteome-wide Domain-centric Perspective on Protein Phosphorylation

Phosphorylation is a widespread post-translational modification that modulates the function of a large number of proteins. Here we show that a significant proportion of all the domains in the human proteome is significantly enriched or depleted in phosphorylation events. A substantial improvement in phosphosites prediction is achieved by leveraging this observation, which has not been tapped by existing methods. Phosphorylation sites are often not shared between multiple occurrences of the same domain in the proteome, even when the phosphoacceptor residue is conserved. This is partly because of different functional constraints acting on the same domain in different protein contexts. Moreover, by augmenting domain alignments with structural information, we were able to provide direct evidence that phosphosites in protein-protein interfaces need not be positionally conserved, likely because they can modulate interactions simply by sitting in the same general surface area.Phosphorylation, the most widespread protein post-translational modification, is an important regulator of protein function. The addition of phosphate groups on serine, threonine, and tyrosine residues can modulate the activity of the target protein by inducing complex conformational changes, by modifying protein electrostatics, and by regulating domain-peptide interactions, as in 14-3-3 or SH2 domains, that specifically recognize phosphorylated residues. The standard experimental technique for the high-throughput identification of phosphorylation sites is mass spectrometry (1).Phosphorylation is catalyzed by protein kinases, a family that in humans comprises ∼540 members (2, 3). It is well understood that these enzymes recognize specific sequence motifs in their substrates (4, 5). Accordingly the sequence around the phosphorylation site is undisputedly the most important feature for phosphosite prediction (6, 7). However the “context,” in a broad sense, where these motifs occur is also important as sequence alone is not enough to achieve the observed specificity of phosphorylation. Therefore, several studies have characterized multiple aspects of phosphosites such as their preference for loops and disordered regions (reviewed in (8)), or the tendency of phosphoserines and phosphothreonines to occur in clusters (9), and these features have been used to improve the performance of phosphosite predictors (6, 7, 10–12). Moreover placing kinases and substrates in the context of protein interaction networks has been shown to improve the prediction of phosphorylation by specific kinases (13).Perhaps one of the most puzzling observations when looking at the phosphoproteome as a whole, is the fact that a large proportion of phosphorylation sites is poorly conserved. This has led to various hypotheses. First some sites may represent nonfunctional, possibly low-stoichiometry, phosphorylation events that are picked up because of the sensitivity of mass-spectrometry (14, 15). Indeed functionally characterized sites and those matching known kinase motifs are more conserved on average (15–17). However, although in biology function often equates with conservation, there could be genuinely functional fast-evolving phosphosites, that are responsible for species-specific differences in signaling and regulation. Moreover in some cases, especially in the regulation of protein-protein interactions, the exact position of the phosphosites may be unimportant (18, 19).Here we explore the issues of “context” and “conservation” of phosphorylation sites from the perspective of protein domains. To this end, we assembled a comprehensive database of phosphosites from publicly available sources and studied their proteome distribution with respect to the location and identity of protein domains. We focus on the human phosphoproteome because it has been very well characterized in a multitude of low- and high-throughput experiments, thus providing the opportunity for a comprehensive, proteome-wide, study. In particular, the issues we want to address are the following:

1. Are specific domain types preferentially phosphorylated? Or conversely are some domains specifically depleted of phosphorylation sites?
2. Can the domain context be used to improve the prediction of phosphorylation sites?
3. What is the conservation pattern of phosphosites when looking at multiple instances of the same domain in the proteome?

     

The Claudin Megatrachea Protein Complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.