高尔基体磷酸化蛋白GOLPH2的研究及其潜在的应用价值

高尔基体磷酸化蛋白（Golgi phosphoprotein2,GOLPH2又称GP73）是一种11型高尔基体跨膜糖蛋白,与肝炎、肝硬化和肝癌等的发生发展密切相关。GP73是一个可靠的诊断HCC及监测复发的血清标记物,其敏感性和特异性均高于传统的AFP。GP73在目前的肝癌早期诊断和治疗中的显示出重要的作用,有可能成为临床上新的肝癌血清标记物。而且部分研究发现GP73对肝癌的转移潜能和预后有重要的预测价值,抑制其表达可显著降低肝癌细胞的恶性表型,这似乎提示GP73可以成为肝癌治疗的新靶点。     

Diagnostic Value of Immunohistochemical Staining of GP73, GPC3, DCP,CD34, CD31, and Reticulin Staining in Hepatocellular Carcinoma

It has been reported that Golgi protein-73 (GP73), glypican-3 (GPC3), and des-γ-carboxy prothrombin (DCP) could serve as serum markers for the early detection of hepatocellular carcinoma (HCC). This study aimed to evaluate a panel of immunostaining markers (including GP73, GPC3, DCP, CD34, and CD31) as well as reticulin staining to distinguish HCC from the mimickers. Our results revealed that CD34 immunostaining and reticulin staining were highly sensitive for the diagnosis of HCC. A special immunoreaction pattern of GP73—a diffuse coarse-block pattern in a perinuclear region or a concentrated cluster-like or cord-like pattern in a certain part of the cytoplasm—was observed in HCC cells, in contrast to the cytoplasmic fine-granular pattern in surrounding non-tumor cells and non-malignant nodules. This coarse-block pattern correlated significantly with less differentiated HCC. In comparison, GPC3 displayed a good advantage in diagnosing well-differentiated HCC. In our study, DCP and CD31 showed little diagnostic value for HCC as an immunostaining marker. When GP73, GPC3, and CD34 were combined, the specificity improved to 96.6%. Our findings demonstrate for the first time that the immunohistochemical panel of GP73, GPC3, and CD34 as well as reticulin staining is highly specific for the pathological diagnosis of HCC.     

Knockdown of Golgi phosphoprotein 73 blocks the trafficking of matrix metalloproteinase‐2 in hepatocellular carcinoma cells and inhibits cell invasion

Golgi phosphoprotein 73 (GP73) has been regarded as a novel serum biomarker for the diagnosis of hepatocellular carcinoma (HCC) in recent years. It has been reported that the upregulation of GP73 may promote the carcinogenesis and metastasis of HCC; however, the mechanisms remain poorly understood. In this study, GP73 correlates positively with matrix metalloproteinase‐2 (MMP‐2) in HCC‐related cells and tissues. Further studies indicate that the knockdown of GP73 blocks MMP‐2 trafficking and secretion, resulting in cell invasion inhibition. Additionally, the knockdown of GP73 induces the accumulation of intracellular MMP‐2, which inhibits the phosphorylation of Src at Y416 and triggers the inhibition of SAPK/JNK and p53‐p21 signalling pathways through a negative feedback loop. Finally, the transactivation of MMP2 was inhibited by the reduction in E2F1. This study reveals that GP73 plays functional roles in the trafficking and equilibrium of epithelial‐mesenchymal transition (EMT)‐related secretory proteins and that GP73 serves as a new potential target for combating the metastasis of HCC.     

Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption

The cis-Golgi protein GPP130 reversibly redistributes to endosomes upon pH disruption, but the identity of the endosomes and the involved cycling route are unknown. It is also unknown whether any other early Golgi proteins participate in this pathway. Here, we analyze GPP130 and the structurally related Golgi protein GP73. Unlike the TGN marker TGN38/46, GPP130 and GP73 colocalized in the early Golgi and redistributed to the ER after brefeldin A treatment. Nevertheless, after pH disruption by monensin, GPP130 and GP73 redistributed to endosomes containing redistributed TGN38/46, but not other endosomal markers. In common with TGN38/46, the redistribution involved transient appearance on the plasma membrane, and upon monensin washout, the proteins moved back to the Golgi along a microtubule- and PI3 kinase-independent route. Although GP73 did not associate with GPP130, its steady-state Golgi targeting was also mediated by a lumenal predicted coiled-coil stem domain. These findings indicate that at least two early Golgi proteins, each containing stem domain Golgi targeting determinants, cycle to the cell surface and back along the late endosome independent TGN38/46 pathway.     

Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein

Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.     

Diagnostic Value of the Combination of Golgi Protein 73 and Alpha-Fetoprotein in Hepatocellular Carcinoma: A Meta-Analysis

Conflicting results have been widely reported on the use of Golgi protein 73 (GP73) as a serum biomarker for diagnosing hepatocellular carcinoma (HCC). This study evaluated the accuracy of GP73, alpha-fetoprotein (AFP), and GP73 + AFP for diagnosing HCC. The meta-analysis was performed on 11 studies that were selected by means of a comprehensive systematic literature review. Summary diagnostic accuracy, meta-regression analysis for heterogeneity and publication bias, and other statistical analyses were performed using Meta-Disc (version 1.4) and Stata (version 12.0). Pooled sensitivity, specificity, and diagnostic odds ratio were 0.77 (95% CI: 0.75–0.79), 0.91 (95% CI: 0.90–0.92), and 12.49 (95% CI: 4.91–31.79) for GP73; 0.62 (95% CI: 0.60–0.64), 0.84 (95% CI: 0.83–0.85), and 11.61 (95% CI: 8.02–16.81) for AFP; and 0.87 (95% CI: 0.85–0.89), 0.85 (95% CI: 0.84–0.86), and 30.63 (95% CI: 18.10–51.84) for GP73 + AFP. The area under the curve values were 0.86, 0.84, and 0.91 for GP73, AFP, and GP73 + AFP, respectively. These results indicate that for HCC diagnosis, the accuracy of GP73 was higher than that of AFP, and that GP73 + AFP exhibited significantly higher diagnostic accuracy than did GP73 or AFP alone.     

The Predictive Value of Golgi Protein 73 in Differentiating Benign from Malignant Liver Tumors

Introduction

In the work up of primary solid liver lesions it is essential to differentiate correctly between benign and malignant tumors, such as hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) respectively. A promising new marker to detect HCC is Golgi Protein 73 (GP73). Studies comparing patients with HCC and cirrhosis with normal controls suggested that GP73 is specific for patients with HCC; however, patients with other liver tumors were not included. We therefore studied the predictive value of GP73 in differentiating between solid benign and malignant liver tumors.

Materials and Methods

This study included 264 patients: 88 patients with HCC, 88 with hepatocellular adenoma (HCA), and 88 with focal nodal hyperplasia (FNH). A blood sample was collected from each patient to measure GP73 levels using a quantitative ELISA assay and differences in outcome between subgroups were compared. The receiver operating characteristic (ROC) curve, sensitivity and specificity of GP73 were calculated and compared to alpha-fetoprotein (AFP) levels.

Results

When comparing malignant and benign liver tumors the area under ROC was 0.701 and 0.912 for GP73 and AFP respectively. Test characteristics revealed a sensitivity of 60% for GP73 and 65% for AFP; in addition the specificity was 77% for GP73 and 96% for AFP.

Conclusion

Although the literature suggests that GP73 is a valuable serum marker in patients with HCC, the serum concentration may also be increased in patients with solid benign liver tumors. Therefore, a GP73 assay is less suitable for discriminating between primary malignant and benign tumors of the liver.     

GP73 与消化系统疾病的研究进展

高尔基体是一个非常重要的细胞器,最近研究表明,它除了蛋白加工外,还能参与细胞分化、细胞间信号传导和细胞凋亡, 其功能障碍也许和疾病的发生、发展有着某种联系。随着蛋白组学技术的发展,使得筛选新的肿瘤标志物成为可能,其中高尔基 体蛋白73（GP73）被认为是最值得期待的血清标志物之一,尤其是早期肝癌的血清标志物。本文对近年来有关GP73 结构、表达分 布以与及消化系统疾病的研究进展进行综述。     

GP73与消化系统疾病的研究进展

高尔基体是一个非常重要的细胞器,最近研究表明,它除了蛋白加工外,还能参与细胞分化、细胞间信号传导和细胞凋亡,其功能障碍也许和疾病的发生、发展有着某种联系。随着蛋白组学技术的发展,使得筛选新的肿瘤标志物成为可能,其中高尔基体蛋白73（GP73）被认为是最值得期待的血清标志物之一,尤其是早期肝癌的血清标志物。本文对近年来有关GP73结构、表达分布以与及消化系统疾病的研究进展进行综述。     

Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.     

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.     

Evaluation of circulating Transforming growth factor-beta1, Glypican-3 and Golgi protein-73 mRNAs expression as predictive markers for hepatocellular carcinoma in Egyptian patients

Hepatocellular carcinoma (HCC) incidence is fast-growing especially in countries highly prevalent with viral hepatitis. Its poor prognosis has driven the research toward the discovery of sensitive markers for early detection. We investigated the usefulness of serum Transforming growth factor-beta1 (TGF-β1), Glypican-3 (GPC3), and Golgi protein-73 (GP73) mRNAs as early biomarkers in HCC Egyptian patients chronically infected with hepatitis C virus (HCV) in comparison with serum alpha-fetoprotein (AFP). Using semi-quantitative RT-PCR and densitometry analysis, circulating TGF-β1, GPC3, and GP73 mRNAs expressions were estimated in 15 healthy adults, 15 chronic HCV (CHC) patients and 25 HCC patients. Serum GP73 expression percentage in HCC group was significantly higher than controls (100 vs. 40 %, P ≤ 0.001) and when compared to elevated serum AFP levels (100 vs. 36 %, P ≤ 0.001). TGF-β1 and GP73 expression means were also higher in HCC patients than controls and CHC patients (P < 0.05). GPC3 expression showed higher frequency in CHC patients compared to HCC group (80 vs. 28 %, P = 0.0016). According to the study cutoffs, serum TGF-β1 and GP73 mRNAs showed 60 and 96 % sensitivities for HCC diagnosis with 100 and 95 % specificities, respectively. Furthermore, elevated GP73 mRNA expression levels in early HCC were significantly increased compared to those of TGF-β1 mRNA and to high serum AFP (92.3 vs. 53.8 and 23.1 %; P = 0.03 and 0.0004, respectively). In conclusion, circulating TGF-β1 and GP73 mRNAs could be useful biomarkers for HCV-induced HCC diagnosis. Moreover, serum GP73 mRNA is sensitive for early cancer detection than AFP and TGF-β1 mRNA. However, these results need further validation studies.     

Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases.

The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.     

gamma-Secretase cleavage site specificity differs for intracellular and secretory amyloid beta

The final step in A beta generation is the cleavage of the C-terminal 99 amino acid residues of the amyloid precursor protein by gamma-secretase. gamma-Secretase activity is closely linked to the multi-transmembrane-spanning proteins presenilin 1 and presenilin 2. To elucidate whether the cleavage site specificities of gamma-secretase leading to the formation of secreted and intracellular A beta are identical, we made use of point mutations close to the gamma-cleavage site, known to have a dramatic effect on the 42/40 ratio of secreted A beta. We found that the selected point mutations only marginally influenced the 42/40 ratio of intracellular A beta, suggesting differences in the gamma-secretase cleavage site specificity for the generation of secreted and intracellular A beta. The analysis of the subcellular compartments involved in the generation of intracellular A beta revealed that A beta is not generated in the early secretory pathway in the human SH-SY5Y neuroblastoma cell line. In this study we identified late Golgi compartments to be involved in the generation of intracellular A beta. Moreover, we demonstrate that the presence of processed PS1 is not sufficient to obtain gamma-secretase processing of the truncated amyloid precursor protein construct C99, proposing the existence of an additional factor downstream of the endoplasmic reticulum and early Golgi required for the formation of an active gamma-secretase complex.     

The low pH in trans-Golgi triggers autocatalytic cleavage of pre-alpha -inhibitor heavy chain precursor

Pre-alpha-inhibitor is a plasma protein whose physiological function is still unknown, but in vitro studies suggest that it might be involved in inflammatory reactions. Pre-alpha-inhibitor consists of a 25- and a 75-kDa polypeptide: bikunin and heavy chain 3 (H3), respectively. H3 is synthesized with a 30-kDa C-terminal extension, which is released in the Golgi complex through cleavage between an Asp and a Pro residue. We now provide evidence that this cleavage is triggered by the low pH in the late Golgi and occurs through an intramolecular process. First, incubation in vitro of the H3 precursor (proH3) at pH 6.0 or lower results in rapid cleavage of the protein. Second, the rate of the cleavage reaction does not depend on the concentration of proH3 and is not affected by the presence of various protease inhibitors. Third, raising the pH in organelles of cells producing proH3 abolishes cleavage during secretion. The amino acid residues near the cleavage site of proH3 differ from those of previously described self-cleaving proteins, indicating that the mechanisms of scission are different.     

Golgi protein 73 and its diagnostic value in liver diseases

Golgi protein 73 (GP73, also referred to as Golph 2) with 400 amino acids is a 73 kDa transmembrane glycoprotein typically found in the cis‐Golg complex. It is primarily expressed in epithelial cells, which has been found upregulated in hepatocytes in patients suffering from both viral and non‐viral liver diseases. GP73 has drawn increasing attention for its potential application in the diagnosis of liver diseases such as hepatitis, liver cirrhosis and liver cancer. Herein, we reviewed the discovery history of GP73 and summarized studies by many groups around the world, aiming at understanding its structure, expression, function, detection methods and the relationship between GP73 and liver diseases in various settings.     

2-Chloroadenosine decreases tyrosylprotein sulfotransferase activity in the Golgi apparatus in PC12 cells. Evidence for a novel receptor.

In the present studies, we investigated the activity of tyrosylprotein sulfotransferase (TPST) in the Golgi apparatus of PC12 cells and the regulation of this enzyme by 2-chloroadenosine, an adenosine receptor agonist. Studies employing continuous sucrose gradient and trypsinization of the membranes demonstrate that TPST is located on the luminal side of Golgi apparatus in PC12 cells. Treatment of PC12 cells with 2-chloroadenosine results in a dose-dependent decrease of TPST activity which is observable as early as 3 h after initiation of treatment, maximizes at 24-48 h with continuous exposure, and is readily reversible upon removal of the drug. While forskolin, an agent that directly increases intracellular cAMP, has no effect on TPST activity, 2-chloroadenosine equally suppressed the enzyme activity in both the wild type and a protein kinase A-deficient mutant strain of PC12 cells, indicating that such regulation of TPST activity by 2-chloroadenosine was independent of cAMP-dependent protein phosphorylation. This effect of 2-chloroadenosine can be potentiated by an adenosine uptake blocker dipyridamole but cannot be elicited by other adenosine A1 or A2 receptor agonists, further suggesting that TPST activity in PC12 cells is regulated by 2-chloroadenosine via a novel membrane receptor. Incubation of the cells with cyclo heximide, a protein synthesis inhibitor, also led to a time- and dose-dependent suppression of TPST activity. At concentrations of cycloheximide that produced maximal inhibition (approximately 50%), cotreatment with 2-chloroadenosine did not lead to a further decrease of the TPST activity. These results suggest that the sensitivity of TPST activity to be controlled by protein synthesis provides a mechanism for regulation of its activity by 2-chloroadenosine.