Ubiquitin-binding associated protein 2 regulates KRAS activation and macropinocytosis in pancreatic cancer.

Macropinocytosis supports the metabolic requirement of RAS-transformed pancreatic ductal adenocarcinoma cells (PDACs). However, regulators of RAS-transformation (activation) that lead to macropinocytosis have not been identified. Herein, we report that UBAP2 (ubiquitin-binding associated protein 2), regulates the activation of KRAS and macropinocytosis in pancreatic cancer. We demonstrate that UBAP2 is highly expressed in both pancreatic cancer cell lines and tumor tissues of PDAC patients. The expression of UBAP2 is associated with poor overall survival in several cancers, including PDAC. Silencing UBAP2 decreases the levels of activated KRAS, and inhibits macropinocytosis, and tumor growth in vivo. Using a UBAP2-deletion construct, we demonstrate that the UBA-domain of UBAP2 is critical for the regulation of macropinocytosis and maintaining the levels of activated KRAS. In addition, UBAP2 regulates RAS downstream signaling and helps maintain RAS in the GTP-bound form. However, the exact mechanism by which UBAP2 regulates KRAS activation is unknown and needs further investigation. Thus, UBAP2 may be exploited as a potential therapeutic target to inhibit macropinocytosis and tumor growth in activated KRAS-driven cancers.

KRCC1: A potential therapeutic target in ovarian cancer.

Using a systems biology approach to prioritize potential points of intervention in ovarian cancer, we identified the lysine rich coiled-coil 1 (KRCC1), as a potential target. High-grade serous ovarian cancer patient tumors and cells express significantly higher levels of KRCC1 which correlates with poor overall survival and chemoresistance. We demonstrate that KRCC1 is predominantly present in the chromatin-bound nuclear fraction, interacts with HDAC1, HDAC2, and with the serine-threonine phosphatase PP1CC. Silencing KRCC1 inhibits cellular plasticity, invasive properties, and potentiates apoptosis resulting in reduced tumor growth. These phenotypes are associated with increased acetylation of histones and with increased phosphorylation of H2AX and CHK1, suggesting the modulation of transcription and DNA damage that may be mediated by the action of HDAC and PP1CC, respectively. Hence, we address an urgent need to develop new targets in cancer.

Gold Nanoparticles sensitize pancreatic cancer cells to gemcitabine.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid cancers with dismal prognosis. Several mechanisms that are mainly responsible for aggressiveness and therapy resistance of PDAC cells include epithelial to mesenchymal transition (EMT), stemness and Mitogen Activated Protein Kinase (MAPK) signaling. Strategies that inhibit these mechanisms are critically important to improve therapeutic outcome in PDAC. In the current study, we wanted to investigate whether gold nanoparticles (AuNPs) could sensitize pancreatic cancer cells to the chemotherapeutic agent gemcitabine. We demonstrated that treatment with AuNPs of 20 nm diameter inhibited migration and colony forming ability of pancreatic cancer cells. Pre-treatment with AuNPs sensitized pancreatic cancer cells to gemcitabine in both viability and colony forming assays. Mechanistically, pre-treatment of pancreatic cancer cells with AuNPs decreased gemcitabine induced EMT, stemness and MAPK activation. Taken together, these findings suggest that AuNPs could be considered as a potential agent to sensitize pancreatic cancer cells to gemcitabine.

Type Iγ phosphatidylinositol phosphate kinase dependent cell migration and invasion are dispensable for tumor metastasis.

Type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) has been associated with poor prognosis in breast cancer patients by promoting metastasis. Among the six alternative-splicing isoforms of PIPKIγ, PIPKIγ_i2 specifically targets to focal adhesions and regulates focal adhesion turnover, thus was proposed responsible for tumor metastasis. In the present study, we specifically depleted PIPKIγ_i2 from mouse triple negative breast cancer (TNBC) 4T1 cells and analyzed their behaviors. As expected, PIPKIγ_i2-depleted 4T1 cells exhibited reduced proliferation, migration, and invasion in vitro at a comparable level as pan-PIPKIγ depleted cells. However, PIPKIγ_i2 depletion had no effect on metastasis and progression of 4T1 tumors in vivo. PIPKIγ_i2-depleted tumors showed similar levels of growth, hypoxia state, macrophage infiltration, and angiogenesis as parental tumors, although the pan-PIPKIγ depletion led to substantial inhibition on these aspects. Further investigation revealed that depleting PIPKIγ_i2 alone, unlike depleting all PIPKIγ isoforms, had no effect on PD-L1 expression, the status of the epithelial-to-mesenchymal transition, and the anchorage-independent growth of 4T1 cells. In human TNBC MDA-MB-231 cells, we obtained similar results. Thus, while PIPKIγ_i2 indeed is required for cell migration and invasion, these characteristics are not decisive for metastasis. Other PIPKIγ isoform(s) that regulate the expression of key factors to support cell survival under stresses is more critical for the malignant progression of TNBCs.

Gold Nanoparticles Disrupt Tumor Microenvironment - Endothelial Cell Cross Talk To Inhibit Angiogenic Phenotypes in Vitro.

It is currently recognized that perpetual cross talk among key players in tumor microenvironment such as cancer cells (CCs), cancer associated fibroblasts (CAFs), and endothelial cells (ECs) plays a critical role in tumor progression, metastasis, and therapy resistance. Disruption of the cross talk may be useful to improve the outcome of therapeutics for which limited options are available. In the current study we investigate the use of gold nanoparticles (AuNPs) as a therapeutic tool to disrupt the multicellular cross talk within the TME cells with an emphasis on inhibiting angiogenesis. We demonstrate here that AuNPs disrupt signal transduction from TME cells (CCs, CAFs, and ECs) to ECs and inhibit angiogenic phenotypes in vitro. We show that conditioned media (CM) from ovarian CCs, CAFs, or ECs themselves induce tube formation and migration of ECs in vitro. Migration of ECs is also induced when ECs are cocultured with CCs, CAFs, or ECs. In contrast, CM from the cells treated with AuNPs or cocultured cells pretreated with AuNPs demonstrate diminished effects on ECs tube formation and migration. Mechanistically, AuNPs deplete ∼95% VEGF165 from VEGF single-protein solution and remove up to ∼45% of VEGF165 from CM, which is reflected on reduced activation of VEGF-Receptor 2 (VEGFR2) as compared to control CM. These results demonstrate that AuNPs inhibit angiogenesis via blockade of VEGF-VEGFR2 signaling from TME cells to endothelial cells.

Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Deregulation of mitochondrial morphogenesis, a dynamic equilibrium between mitochondrial fusion and fission processes, is now evolving as a key metabolic event that fuels tumor growth and therapy resistance. However, fundamental knowledge underpinning how cancer cells reprogram mitochondrial morphogenesis remains incomplete. Here, we report that cystathionine ß-synthase (CBS) reprograms mitochondrial morphogenesis in ovarian cancer (OvCa) cells by selectively regulating the stability of mitofusin 2 (MFN2). Clinically, high expression of both CBS and MFN2 implicates poor overall survival of OvCa patients, and a significant association between CBS and MFN2 expression exists in individual patients in the same data set. The silencing of CBS by small interfering RNA or inhibition of its catalytic activity by a small molecule inhibitor creates oxidative stress that activates JNK. Activated JNK phosphorylates MFN2 to recruit homologous to the E6-AP carboxyl terminus' domain-containing ubiquitin E3 ligase for its degradation via the ubiquitin-proteasome system. Supplementation with hydrogen sulfide or glutathione (the catalytic products of CBS enzymatic activity), anti-oxidants, or a JNK inhibitor restores MFN2 expression. In CBS-silenced orthotopic xenograft tumor tissues, MFN2 but not MFN1 is selectively downregulated. In summary, this report reveals a role for deregulated mitochondrial morphogenesis in OvCa, suggests one of the mechanisms for this deregulation, and provides a way to correct it through modulation of the metabolic enzyme CBS.-Chakraborty, P. K., Murphy, B., Mustafi, S. B., Dey, A., Xiong, X., Rao, G., Naz, S., Zhang, M., Yang, D., Dhanasekaran, D. N., Bhattacharya, R., Mukherjee, P. Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Evaluating the Mechanism and Therapeutic Potential of PTC-028, a Novel Inhibitor of BMI-1 Function in Ovarian Cancer.

BMI-1, also known as a stem cell factor, is frequently upregulated in several malignancies. Elevated expression of BMI-1 correlates with poor prognosis and is therefore considered a viable therapeutic target in a number of malignancies including ovarian cancer. Realizing the immense pathologic significance of BMI-1, small-molecule inhibitors against BMI-1 are recently being developed. In this study, we functionally characterize PTC-028, an orally bioavailable compound that decreases BMI-1 levels by posttranslational modification. We report that PTC-028 treatment selectively inhibits cancer cells in clonal growth and viability assays, whereas normal cells remain unaffected. Mechanistically, hyperphosphorylation-mediated depletion of cellular BMI-1 by PTC-028 coupled with a concurrent temporal decrease in ATP and a compromised mitochondrial redox balance potentiates caspase-dependent apoptosis. In vivo, orally administered PTC-028, as a single agent, exhibits significant antitumor activity comparable with the standard cisplatin/paclitaxel therapy in an orthotopic mouse model of ovarian cancer. Thus, PTC-028 has the potential to be used as an effective therapeutic agent in patients with epithelial ovarian cancer, where treatment options are limited. Mol Cancer Ther; 17(1); 39-49. ©2017 AACR.

MICU1 drives glycolysis and chemoresistance in ovarian cancer.

Cancer cells actively promote aerobic glycolysis to sustain their metabolic requirements through mechanisms not always clear. Here, we demonstrate that the gatekeeper of mitochondrial Ca2+ uptake, Mitochondrial Calcium Uptake 1 (MICU1/CBARA1) drives aerobic glycolysis in ovarian cancer. We show that MICU1 is overexpressed in a panel of ovarian cancer cell lines and that MICU1 overexpression correlates with poor overall survival (OS). Silencing MICU1 in vitro increases oxygen consumption, decreases lactate production, inhibits clonal growth, migration and invasion of ovarian cancer cells, whereas silencing in vivo inhibits tumour growth, increases cisplatin efficacy and OS. Mechanistically, silencing MICU1 activates pyruvate dehydrogenase (PDH) by stimulating the PDPhosphatase-phosphoPDH-PDH axis. Forced-expression of MICU1 in normal cells phenocopies the metabolic aberrations of malignant cells. Consistent with the in vitro and in vivo findings we observe a significant correlation between MICU1 and pPDH (inactive form of PDH) expression with poor prognosis. Thus, MICU1 could serve as an important therapeutic target to normalize metabolic aberrations responsible for poor prognosis in ovarian cancer.

EGFR-induced phosphorylation of type Iγ phosphatidylinositol phosphate kinase promotes pancreatic cancer progression.

Pancreatic cancer is one of the deadliest malignancies and effective treatment has always been lacking. In current study, we investigated how the type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) participates in the progression of pancreatic ductal adenocarcinoma (PDAC) for novel therapeutic potentials against this lethal disease. We found that PIPKIγ is up-regulated in all tested PDAC cell lines. The growth factor (including EGFR)-induced tyrosine phosphorylation of PIPKIγ is significantly elevated in in situ and metastatic PDAC tissues. Loss of PIPKIγ inhibits the aggressiveness of PDAC cells by restraining the activities of AKT and STAT3, as well as MT1-MMP expression. Therefore when planted into the pancreas of nude mice, PIPKIγ-depleted PDAC cells exhibits substantially repressed tumor growth and metastasis comparing to control PDAC cells. Results from further studies showed that the phosphorylation-deficient PIPKIγ mutant, unlike its wild-type counterpart, cannot rescue PDAC progression inhibited by PIPKIγ depletion. These findings indicate that PIPKIγ, functioning downstream of EGFR signaling, is critical to the progression of PDAC, and suggest that PIPKIγ is potentially a valuable therapeutic target for PDAC treatment.

Gold Nanoparticle Reprograms Pancreatic Tumor Microenvironment and Inhibits Tumor Growth.

Altered tumor microenvironment (TME) arising from a bidirectional crosstalk between the pancreatic cancer cells (PCCs) and the pancreatic stellate cells (PSCs) is implicated in the dismal prognosis in pancreatic ductal adenocarcinoma (PDAC), yet effective strategies to disrupt the crosstalk is lacking. Here, we demonstrate that gold nanoparticles (AuNPs) inhibit proliferation and migration of both PCCs and PSCs by disrupting the bidirectional communication via alteration of the cell secretome. Analyzing the key proteins identified from a functional network of AuNP-altered secretome in PCCs and PSCs, we demonstrate that AuNPs impair secretions of major hub node proteins in both cell types and transform activated PSCs toward a lipid-rich quiescent phenotype. By reducing activation of PSCs, AuNPs inhibit matrix deposition, enhance angiogenesis, and inhibit tumor growth in an orthotopic co-implantation model in vivo. Auto- and heteroregulations of secretory growth factors/cytokines are disrupted by AuNPs resulting in reprogramming of the TME. By utilizing a kinase dead mutant of IRE1-α, we demonstrate that AuNPs alter the cellular secretome through the ER-stress-regulated IRE1-dependent decay pathway (RIDD) and identify endostatin and matrix metalloproteinase 9 as putative RIDD targets. Thus, AuNPs could potentially be utilized as a tool to effectively interrogate bidirectional communications in the tumor microenvironment, reprogram it, and inhibit tumor growth by its therapeutic function.

Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Deficiencies of the human cystathionine ß-synthase (CBS) enzyme are characterized by a plethora of vascular disorders and hyperhomocysteinemia. However, several clinical trials demonstrated that despite reduction in homocysteine levels, disease outcome remained unaffected, thus the mechanism of endothelial dysfunction is poorly defined. Here, we show that the loss of CBS function in endothelial cells (ECs) leads to a significant down-regulation of cellular hydrogen sulfide (H2S) by 50% and of glutathione (GSH) by 40%. Silencing CBS in ECs compromised phenotypic and signaling responses to the VEGF that were potentiated by decreased transcription of VEGF receptor (VEGFR)-2 and neuropilin (NRP)-1, the primary receptors regulating endothelial function. Transcriptional down-regulation of VEGFR-2 and NRP-1 was mediated by a lack in stability of the transcription factor specificity protein 1 (Sp1), which is a sulfhydration target of H2S at residues Cys68 and Cys755. Reinstating H2S but not GSH in CBS-silenced ECs restored Sp1 levels and its binding to the VEGFR-2 promoter and VEGFR-2, NRP-1 expression, VEGF-dependent proliferation, and migration phenotypes. Thus, our study emphasizes the importance of CBS-mediated protein S-sulfhydration in maintaining vascular health and function.-Saha, S., Chakraborty, P. K., Xiong, X., Dwivedi, S. K. D., Mustafi, S. B., Leigh, N. R., Ramchandran, R., Mukherjee, P., Bhattacharya, R. Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Role of cystathionine beta synthase in lipid metabolism in ovarian cancer.

Elevated lipid metabolism is implicated in poor survival in ovarian cancer (OC) and other cancers; however, current lipogenesis-targeting strategies lack cancer cell specificity. Here, we identify a novel role of cystathionine beta-synthase (CBS), a sulphur amino acid metabolizing enzyme highly expressed in several ovarian cancer cell lines, in driving deregulated lipid metabolism in OC. We examined the role of CBS in regulation of triglycerides, cholesterol and lipogenic enzymes via the lipogenic transcription factors SREBP1 and SREBP2. CBS silencing attenuated the expression of number of key enzymes involved in lipid synthesis (FASN and ACC1). Additionally CBS abrogates lipid uptake in OC cells. Gene silencing of CBS or SREBPs abrogated cellular migration and invasion in OC, while ectopic expression of SREBPs can rescue phenotypic effects of CBS silencing by restoring cell migration and invasion. Mechanistically, CBS represses SREBP1 and SREBP2 at the transcription levels by modulating the transcription factor Sp1. We further established the roles of both CBS and SREBPs in regulating ovarian tumor growth in vivo. In orthotopic tumor models, CBS or SREBP silencing resulted in reduced tumor cells proliferation, blood vessels formation and lipid content. Hence, cancer-selective disruption of the lipid metabolism pathway is possible by targeting CBS and, at least for OC, promises a profound benefit.

Sensitization of ovarian cancer cells to cisplatin by gold nanoparticles.

Recently we reported that gold nanoparticles (AuNPs) inhibit ovarian tumor growth and metastasis in mice by reversing epithelial-mesenchymal transition (EMT). Since EMT is known to confer drug resistance to cancer cells, we wanted to investigate whether anti-EMT property of AuNP could be utilized to sensitize ovarian cancer cells to cisplatin. Herein, we report that AuNPs prevent cisplatin-induced acquired chemoresistance and stemness in ovarian cancer cells and sensitize them to cisplatin. AuNPs inhibit cisplatin induced EMT, decrease the side population cells and key stem cell markers such as ALDH1, CD44, CD133, Sox2, MDR1 and ABCG2 in ovarian cancer cells. Mechanistically, AuNPs prevent cisplatin-induced activation of Akt and NF-kB signaling axis in ovarian cancer cells that are critical for EMT, stem cell maintenance and drug resistance. In vivo, AuNPs sensitize orthotopically implanted ovarian tumor to a low dose of cisplatin and significantly inhibit tumor growth via facilitated delivery of both AuNP and cisplatin. These findings suggest that by depleting stem cell pools and inhibiting key molecular pathways gold nanoparticles sensitize ovarian cancer cells to cisplatin and may be used in combination to inhibit tumor growth and metastasis in ovarian cancer.

PIPKIγ targets to the centrosome and restrains centriole duplication.

Centriole biogenesis depends on the polo-like kinase (PLK4) and a small group of structural proteins. The spatiotemporal regulation of these proteins at pre-existing centrioles is essential to ensure that centriole duplication occurs once per cell cycle. Here, we report that phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C, hereafter referred to as PIPKIγ) plays an important role in centriole fidelity. PIPKIγ localized in a ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S and G2 phases, but not in M phase. This localization was dependent upon an association with centrosomal protein of 152 KDa (CEP152). Without detaining cells in S or M phase, the depletion of PIPKIγ led to centriole amplification in a manner that was dependent upon PLK4 and spindle assembly abnormal protein 6 homolog (SAS6). The expression of exogenous PIPKIγ reduced centriole amplification that occurred as a result of endogenous PIPKIγ depletion, hydroxyurea treatment or PLK4 overexpression, suggesting that PIPKIγ is likely to function at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic polo-box domain of PLK4 and that this binding reduced the kinase activity of PLK4. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication, which acts by modulating the homeostasis of PLK4 activity.

An association between type Iγ PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization.

E-Cadherin-mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P(2) is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P(2) pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.

Cloning and characterization of an IKK homologue from pearl oyster, Pinctada fucata.

IkappaB kinase (IKK) play central roles in cell signaling by regulating nuclear factor-kappaB (NF-kappaB) activation, which is involved in inflammatory response, proliferation, development and bone homeostasis. We report here for the first time that an IKK homologue was cloned and functionally characterized in pearl oyster, Pinctada fucata. The full-length cDNA consists of 2546bp with an ORF encoding a 737 amino acids protein. The putative pearl oyster IKK protein (Pf-IKK) possesses the characteristic organization of the mammalian IKK proteins, namely an amino-terminal kinase domain followed by a leucine zipper region and a carboxylterminal helix-loop-helix motif. Real-time PCR (RT-PCR) analysis indicated that Pf-IKK was ubiquitously expressed in pearl oyster. We also found that lipopolysaccharides (LPS) transiently stimulates IkappaBalpha degradation, but not expression levels of Pf-IKK. When transfected into NIH3T3 cells, Pf-IKK activated the expression of NF-kappaB-controlled reporter gene and induced NF-kappaB translocation, whereas the activation was greatly deduced by pyrrolidine dithiocarbamate (PDTC). We also found that overexpression of Pf-IKK increased the alkaline phosphatase (ALP) activity significantly. Based on the results and the homology to the vertebrate NF-kappaB cascade, these studies help to highlight a potentially important regulatory pathway to the study of the related functions in mollusks.

Cloning and characterization of a heterogeneous nuclear ribonucleoprotein homolog from pearl oyster, Pinctada fucata.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) have fundamental roles in the post-transcriptional control of gene expression. Here, a cDNA encoding a presumed full-length RNA-binding protein was isolated from pearl oyster (Pinctada fucata) using reverse transcription-polymerase chain reaction with degenerate primers, and rapid amplification of cDNA ends. The full-length cDNA consists of 2737 bp with an open reading frame encoding a protein of 624 amino acids with a predicted molecular weight of 69 kDa and isoelectric point of 8.7. The putative pearl oyster RNA-binding protein presents a molecular organization close to the hnRNPs, namely an acidic N-terminal followed by three RNA-recognition motifs and a C-terminal that contains RG/RGG repeated motifs. When transfected HeLa cells, the Pf-HRPH (Pinctada fucata hnRNP homolog) gene expression product was found only in nuclei, revealing that it is a nuclear protein. The expression pattern was also investigated by quantitative real-time polymerase chain reaction, indicating that Pf-HRPH mRNA was abundantly expressed in gonad, gill, and viscera. As far as we know, the putative Pf-HRPH is the first hnRNP homolog cloned in mollusks. These data are significant for further study of the multiple functions of RNA-binding protein.

Pf-Rel, a Rel/nuclear factor-kappaB homolog identified from the pearl oyster, Pinctada fucata.

Transcription factor Rel/nuclear factor-kappa B (NF-kappaB) has been the focus of many studies since its discovery in 1986. Different homologs of Rel/NF-kappaB have been found in both vertebrate and invertebrate. A cDNA clone encoding a putative Rel/NF-kappaB homolog (designated Pf-Rel) was isolated from the pearl oyster, Pinctada fucata. The sequence of Pf-Rel consists of the Rel homology domain, IPT NF-kappaB domain and C-terminal transactivation domain. Sequence analysis of Pf-Rel shows that it shares high similarity with other Rel/NF-kappaB family proteins, especially within the conserved domains. Reverse transcription-polymerase chain reaction analysis revealed that Pf-Rel mRNA was expressed ubiquitously. Further in situ hybridization analysis showed that Pf-Rel mRNA was expressed mainly at the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold. The identification and characterization of pearl oyster Pf-Rel help to further investigate the involvement of Rel/NF-kappaB in oyster immunity and other biological processes.

Pf-ALMP, a novel astacin-like metalloproteinase with cysteine arrays, is abundant in hemocytes of pearl oyster Pinctada fucata.

The astacin family metalloproteinase is a family of zinc-dependent endopeptidases which play crucial roles in embryonic development, bone growth and morphogenesis. A cDNA clone encoding a putative astacin-like metalloproteinase (pf-ALMP) was isolated from hemocytes of pearl oyster, Pinctada fucata. The novel metalloproteinase presents a molecular organization close to the astacins, but has a novel C-terminal domain with cysteine arrays. RT-PCR analysis revealed that pf-ALMP was expressed dramatically high in hemocytes, which was affected by lipopolysaccharides (LPS) challenge. High expression of pf-ALMP was also found in gill, gonad and digestion gland, and in situ hybridization demonstrated that pf-ALMP was expressed in the epithelia cells of these tissues. Substrate analysis studies indicated that the recombinant pf-ALMP catalytic domain could digest gelatin. Interestingly, the pf-ALMP also could be involved in cell proliferation processes and the cysteine arrays were necessary for the proliferative activity. Taken together, these studies also help to further understand the functions of astacins which may be related to the processes of molluscan inflammatory response, embryo development, proliferation and shell formation.

Cloning and characterization of a novel G protein beta-subunit of pearl oyster (Pinctada fucata), and its interaction sites with calmodulin.

A cDNA clone encoding a novel G protein beta subunit of beta1 subclass, pfGbeta1 was isolated from the pearl oyster (Pinctada fucata). The deduced amino acid sequence of pfGbeta1 (341 amino acids) shares high homology to northern European squid (Loligo pealei) and great pond snail (Lymnaea stagnalis) pfGbeta1, while it has diverged from bovine (Bos taurus) and human. The well-conserved amino acid domains in G protein beta subunit, seven WD repeats, were founded in the deduced amino acid sequence. Alignment analysis showed that the beginning amino acid residues in variable fragment of the seventh WD motif are different from any other Gbeta. The prediction of 3D structure of pfGbeta showed that pfGbeta belongs to beta-propeller family proteins whose members contain 4-8 antiparallel beta-sheets resembling the blades of a propeller. In situ hybridization and Northern blotting analysis revealed that the pfGbeta mRNA hybridization signals were widely expressed in various tissues except muscle, with abundantly in epithelia of gill, gonad and outer fold of mantle. We also investigated the interactions between Gbetagamma and calmodulin (CaM), and specific amino acid residues that may be critical for the binding of Gbetagamma to CaM were also identified. Furthermore, the functional studies of the interaction showed that the binding of CaM and Gbetagamma increases the alkaline phosphatase (ALP) activity, an indicator for mineralization in MC3T3-E1 cells. The ALP activity of the mutants of pfGbetagamma that impaired the interactions of Gbetagamma with CaM is higher than the Control group; however, it is lower than the WTC group. Together, these results suggest that the Gbetagamma might interact with CaM and point to the important physiological function in modulating cellular functions.