[Value of podocalyxin levels in urinary extracellular vesicles for diagnosis of diabetic nephropathy].

OBJECTIVE: To explore the value of detecting podocalyxin (PCX) level in urinary extracellular vesicles for the diagnosis of diabetic nephropathy. METHODS: This study was conducted among 57 diabetic patients admitted during the period from March to September, 2017, including 34 with uncomplicated diabetics and 23 with diabetic nephropathy; 21 patients with other types of nephropathy and 11 healthy individuals were also included to serve as the controls. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to verify the separation of urinary extracellular vesicles. The molecular markers of extracellular vesicles (TSG101 and podocalyxin [PCX]) were detected using Western blotting. PCX levels in extracellular vesicles were also detected using ELISA. RESULTS: TEM reveal the presence of numerous extracellular vesicles in the urine with intact morphology and different sizes, and most of them were below 300 nm in diameter as shown by NTA. TSG101 expression was detected in the samples from all the 4 groups. Positive expression of PCX was detected in the samples from patients with diabetic nephropathy but not in the other groups. In patients with diabetic nephropathy, the mean PCX levels (3.27±2.30 ng/µmol)was significantly higher than those in the healthy control group (1.22±0.36 ng/µmol), uncomplicated diabetes group (2.22±1.29 ng/µmol) and nephropathy group (1.24±0.45 ng/µmol). CONCLUSIONS: PCX level in urinary extracellular vesicles is significantly increased in patients with diabetic nephropathy, suggesting the value of PCX as a potential marker for clinical diagnosis of diabetic nephropathy.

VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

ALIX Regulates the Ubiquitin-Independent Lysosomal Sorting of the P2Y1 Purinergic Receptor via a YPX3L Motif.

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination. Here, we describe an ubiquitin-independent lysosomal sorting pathway for the purinergic GPCR P2Y1. After activation, P2Y1 sorts to lysosomes for degradation independent of direct ubiquitination that is mediated by a YPX3L motif within the second intracellular loop that serves as a binding site for the adaptor protein ALIX. Depletion of ALIX or site-directed mutation of the YPX3L motif inhibits P2Y1 sorting into the lumen of multivesicular endosomes/lysosomes and degradation. These findings confirm the function of YPX3L motifs as lysosomal targeting sequences for GPCRs and demonstrate that ALIX mediates the ubiquitin-independent degradation of certain GPCRs.

Exosomes in colorectal carcinoma formation: ALIX under the magnifying glass.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 µm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

Nedd4L expression is decreased in ovarian epithelial cancer tissues compared to ovarian non-cancer tissue.

AIM: Recent studies have demonstrated that the neural precursor cell expressed, developmentally downregulated 4-like (Nedd4L) gene plays a role in the progression of various cancers. However, reports describing Nedd4L expression in ovarian cancer tissues are limited. MATERIAL AND METHODS: A cohort (n = 117) of archival formalin-fixed, paraffin embedded resected normal ovarian epithelial tissues (n = 10), benign ovarian epithelial tumor tissues (n = 10), serous borderline ovarian epithelial tumor tissues (n = 14), mucous borderline ovarian epithelial tumor tissues (n = 11), and invasive ovarian epithelial cancer tissues (n = 72) were assessed for Nedd4L protein expression using immunohistochemistry. RESULTS: Nedd4L protein expression was significantly decreased in invasive ovarian epithelial cancer tissues compared to non-cancer tissues (P < 0.05). Decreased Nedd4L protein expression correlated with clinical stage, pathological grade, lymph node metastasis and survival (P < 0.05). CONCLUSION: Nedd4L protein expression may be an independent prognostic marker of ovarian cancer development.

Immunohistochemical analysis of expressions of RB1, CDK4, HSP90, cPLA2G4A, and CHMP2B is helpful in distinction between myxofibrosarcoma and myxoid liposarcoma.

The role and diagnostic efficacy of gene and protein products RB1, CDK4, CHMP2B, HSP90, and cPLA2G4A, all previously shown to be involved in tumor genesis and cell proliferation, were examined by immunohistochemical techniques in 32 cases of myxofibrosarcomas and 29 myxoid liposarcomas (all diagnosis had been confirmed by fluorescence in situ hybridization). HSP90 demonstrated strong nuclear and cytoplasmic positivity in all myxoid liposarcoma cases, while only 4 myxofibrosarcomas showed scattered HSP90 positivity. All but 4 cases of myxofibrosarcoma displayed strong positivity for cPLA2G4A, while only 2 myxoid liposarcoma cases were cPLA2G4A positive and both were CHMP2B negative. Overexpression of both cPLA2G4A and CHMP2B also suggested higher tumor grade. In conclusion, HSP90 and cPLA2G4A immunohistochemical stains are useful markers to distinguish myxofibrosarcoma from myxoid liposarcoma.

The ESCRT machinery: from the plasma membrane to endosomes and back again.

The manipulation and reorganization of lipid bilayers are required for diverse cellular processes, ranging from organelle biogenesis to cytokinetic abscission, and often involves transient membrane disruption. A set of membrane-associated proteins collectively known as the endosomal sorting complex required for transport (ESCRT) machinery has been implicated in membrane scission steps, which transform a single, continuous bilayer into two distinct bilayers, while simultaneously segregating cargo throughout the process. Components of the ESCRT pathway, which include 5 distinct protein complexes and an array of accessory factors, each serve discrete functions. This review focuses on the molecular mechanisms by which the ESCRT proteins facilitate cargo sequestration and membrane remodeling and highlights their unique roles in cellular homeostasis.

CK2 involvement in ESCRT-III complex phosphorylation.

The multivesicular body (MVB) sorting pathway is a mechanism for delivering transmembrane proteins into the lumen of the lysosome for degradation. ESCRT-III is the final complex in the pathway that assembles on endosomes and executes membrane scission of intraluminal vesicles. In addition, proteins of this complex are involved in other topologically similar processes such as cytokinesis, virus egress and autophagy. Here we show that protein kinase CK2α is involved in the phosphorylation of the ESCRT-III subunits CHMP3 and CHMP2B, as well as of VPS4B/SKD1, an ATPase that mediates ESCRT-III disassembly. This phosphorylation is observed both in vitro and in cells. While we do not observe recruitment of CK2α to endosomes, we demonstrate the localization of CK2α to midbodies during cytokinesis. Phosphomimetic and non-phosphorylatable mutants of ESCRT-III proteins can still bind endosomes and localize to midbodies, indicating that CK2α does not regulate ESCRT-III localization. Finally, we analyzed two cellular functions where CHMP3, CHMP2B and VPS4 are known to be involved, epidermal growth factor degradation and cytokinetic abscission. We demonstrate that the former is impaired by CK2α downregulation whereas the latter is not affected. Taken together, our results indicate that CK2α regulates the function of ESCRT-III proteins in MVB sorting.

Alterations in the levels of vesicular trafficking proteins involved in HIV replication in the brains and CSF of patients with HIV-associated neurocognitive disorders.

Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND) remain prevalent despite improved antiretroviral therapies. A HAND-specific biomarker indicative of neuropsychological impairment (NPI) would give insight into disease progression and aid clinicians in designing therapy. Endosomal sorting complex required for transport (ESCRT) proteins such as tumor susceptibility gene (TSG)-101, vacuolar protein sorting (VPS)-4 and LIP-5 are important for HIV replication and recently antiviral interferon stimulated gene (ISG)-15 was proposed as a biomarker for CNS injury. Here, we analyzed a well-characterized cohort of HIV+ cerebral spinal fluid (CSF) and postmortem brain specimens for multiple vesicular trafficking proteins and a related innate immune protein, ISG-15, TSG-101, VPS-4 and LIP-5. All protein levels trended higher with increased NPI and neuropathology. ISG-15 CSF levels were increased in HIV encephalitis (HIVE) compared to normal cases, and three quarters of HIVE samples had above average CSF ISG-15 levels. VPS-4 CSF levels were increased in NPI/NPI-O compared to normal patients. VPS-4 CSF levels in HIV-associated dementia were equivalent to that of normal patients. LIP-5 CSF levels positively correlate with ISG-15 levels, and higher than average ISG-15 levels indicate elevated viral load. Immunoblot and immunohistochemical analyses show increased expression of ISG-15, VPS-4 and LIP-5 in neuronal cell bodies and astroglial cells. ESCRT protein CSF levels analyzed in conjunction with viral load may be indicative of NPI stage, and may aid in the diagnosis and design of therapies for HIV patients. Further studies on the ESCRT protein expression during HIV infection may lead to a promising biomarker for predicting progression of NPI.

Is NEDD4-1 a negative regulator of phosphatase and tensin homolog in gastric carcinogenesis?

The expression of phosphatase and tensin homolog (PTEN), a tumor suppressor gene, is frequently down-regulated in gastric carcinomas due to mutation, loss of heterozygosity, and promoter hypermethylation. However, it is unknown if additional mechanisms may account for the down-regulation of PTEN expression. While neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) is believed to be a potential dual regulator of PTEN, there are conflicting reports regarding their interaction. To gain further insight into the role of NEDD4-1 and its association with PTEN in gastric carcinoma development, we measured the protein expression of NEDD4-1 and PTEN in gastric mucosae with various pathological lesions and found that NEDD4-1 increased from normal gastric mucosa to intestinal metaplasia and decreased from dysplasia to gastric carcinoma. These changes did not correlate with PTEN expression changes during gastric carcinogenesis. Moreover, we found similar results in protein levels in the primary tumors and adjacent non-tumorous tissues. These results differ from a previous report showing that expression of NEDD4-1 is up-regulated in gastric carcinomas, and show a more complex pattern of NEDD4-1 gene expression during gastric carcinogenesis.

Up-regulation of galectin-3 and Sambucus nigra agglutinin binding site is associated with invasion, metastasis and poor-progression of the gallbladder adenocarcinoma.

BACKGROUND/AIMS: To study the expression of galectin-3 (gal-3) and Sambucus nigra agglutinin (SNA) binding site and to detect their clinicopathological significances in the benign and malignant lesions of gallbladder. METHODOLOGY: We used immunohistochemistry to detect gal-3 expression and ABC affinity-cytochemistry to detect SNA binding site in specimens of adenocarcinoma, peritumoral tissues, polyp and chronic cholecystitis. RESULTS: The positive expression rates of gal-3 and SNA binding site were significantly higher in adenocarcinoma (62.0%, 66.7%) than those in peritumoral tissues (39.1%, 45.6%), polyp (26.7%, 33.3%) and chronic cholecystitis (11.4%, 11.4%) (p<0.05). A high consistency was found between the levels of expression of gal-3 expression and SNA binding site in adenocarcinoma (χ²=9.51, p<0.01). Univariate Kaplan-Meier analysis showed that increased expression of gal-3 (p=0.028) or SNA binding site (p=0.030) was associated with decreased overall survival. Multivariate Cox regression analysis showed that increased expression of gal-3 (p=0.012) or SNA binding site (p=0.030) was an independent prognostic predictor in gallbladder adenocarcinoma. CONCLUSIONS: These results suggest that expression of gal-3 and SNA binding site might have important effects on the carcinogenesis, progression and biological behaviors of gallbladder cancer.

Decreased expression of Nedd4L correlates with poor prognosis in gastric cancer patient.

The neural precursor cell expressed, developmentally down-regulated 4-like (Nedd4L) gene encodes an ubiquitin ligase that targets the epithelial sodium channel for degradation. Recent studies have demonstrated that Nedd4L plays a role in the progression of various cancers. However, the clinical implications of Nedd4L expression status in gastric cancer have remained unclear. We examined the Nedd4L expression in 82 gastric cancer patients by immunohistochemistry. The correlation between Nedd4L expression and clinicopathological factors, including prognosis, was evaluated. Cancerous Nedd4L expression was detectable in 36 of the 82 tumors (43.9%). Tumors with negative Nedd4L expression had greater extent of lymph node metastasis, lymphatic invasion, and venous invasion, and were at a worse stage than the tumors with positive Nedd4L expression. Additionally, the patients with negative Nedd4L expression had poor clinical outcomes. Furthermore, multivariate analysis indicated that Nedd4L expression was an independent prognostic factor for gastric cancer patients. Our results suggest for the first time that negative Nedd4L expression is strongly related to the invasion and metastasis of gastric cancer. Therefore, Nedd4L expression can be used as an independent prognostic marker of gastric cancer.

Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division.

Members of the crenarchaeal kingdom, such as Sulfolobus, divide by binary fission yet lack genes for the otherwise near-ubiquitous tubulin and actin superfamilies of cytoskeletal proteins. Recent work has established that Sulfolobus homologs of the eukaryotic ESCRT-III and Vps4 components of the ESCRT machinery play an important role in Sulfolobus cell division. In eukaryotes, several pathways recruit ESCRT-III proteins to their sites of action. However, the positioning determinants for archaeal ESCRT-III are not known. Here, we identify a protein, CdvA, that is responsible for recruiting Sulfolobus ESCRT-III to membranes. Overexpression of the isolated ESCRT-III domain that interacts with CdvA results in the generation of nucleoid-free cells. Furthermore, CdvA and ESCRT-III synergize to deform archaeal membranes in vitro. The structure of the CdvA/ESCRT-III interface gives insight into the evolution of the more complex and modular eukaryotic ESCRT complex.

Expression and localization of the deubiquitinating enzyme mUBPy in wobbler mouse testis during spermiogenesis.

Mouse ubiquitin-specific processing protease (mUBPy) is a deubiquitinating enzyme highly expressed in both brain and testis. In testis, it interacts with the DnaJ protein, MSJ-1; both mUBPy and MSJ-1 are located on the cytoplasmic surface of the developing acrosome and in the centrosomal region during spemiogenesis. Present data show the first appearance in testis of mUbpy mRNA and protein at 10 days post-partum (d.p.p.). In addition, to investigate on a possible role of mUBPy in sperm formation, we took advantage of mutant wr/wr (wobbler) mice characterized by male infertility, which is likely due to the lack of a real, functional acrosome. RT-PCR and Northern blot analyses show that mUbpy is up-regulated in adult wobbler testis. Furthermore, in wild-type testis mUBPy protein is primarily detected by Western blot in the soluble (cytosolic/nuclear) fraction during the first round of spermatogenesis and in the adult. By contrast, mUBPy is primarily detected in membranous/insoluble protein fraction when wobbler phenotype is clearly shown (30 d.p.p.) and in adult wobbler testis. By immunohistochemistry, whereas in wild-type animals mUBPy marks the profile of the acrosomic vesicle in differentiating spermatids, in wobbler mice only a detergent pre-treatment procedure allows to detect mUBPy immunoreactivity, which results in diffuse spotted granules inside the cytoplasm and around the nuclear shape. In conclusion, in wobbler testis expression of mUbpy is up-regulated, while a differential sorting of the protein characterizes wobbler spermatids where acrosome formation is impaired.