PRKCSH contributes to TNFSF resistance by extending IGF1R half-life and activation in lung cancer.

Tumor necrosis factor superfamily (TNFSF) resistance contributes to the development and progression of tumors and resistance to various cancer therapies. Tumor-intrinsic alterations involved in the adaptation to the TNFSF response remain largely unknown. Here, we demonstrate that protein kinase C substrate 80K-H (PRKCSH) abundance in lung cancers boosts oncogenic IGF1R activation, leading to TNFSF resistance. PRKCSH abundance is correlated with IGF1R upregulation in lung cancer tissues. Specifically, PRKCSH interacts with IGF1R and extends its half-life. The PRKCSH-IGF1R axis in tumor cells impairs caspase-8 activation, increases Mcl-1 expression, and inhibits caspase-9, leading to an imbalance between cell death and survival. PRKCSH deficiency augmented the antitumor effects of natural killer (NK) cells, representative TNFSF effector cells, in a tumor xenograft IL-2Rg-deficient NOD/SCID (NIG) mouse model. Our data suggest that PRKCSH plays a critical role in TNFSF resistance and may be a potential target to improve the efficacy of NK cell-based cancer therapy.

ERVW-1 Activates ATF6-Mediated Unfolded Protein Response by Decreasing GANAB in Recent-Onset Schizophrenia.

Schizophrenia, a mental disorder, afflicts 1% of the worldwide population. The dysregulation of homeostasis in the endoplasmic reticulum (ER) has been implicated in schizophrenia. Moreover, recent studies indicate that ER stress and the unfolded protein response (UPR) are linked to this mental disorder. Our previous research has verified that endogenous retrovirus group W member 1 envelope (ERVW-1), a risk factor for schizophrenia, is elevated in individuals with schizophrenia. Nevertheless, no literature is available regarding the underlying relationship between ER stress and ERVW-1 in schizophrenia. The aim of our research was to investigate the molecular mechanism connecting ER stress and ERVW-1 in schizophrenia. Here, we employed Gene Differential Expression Analysis to predict differentially expressed genes (DEGs) in the human prefrontal cortex of schizophrenic patients and identified aberrant expression of UPR-related genes. Subsequent research indicated that the UPR gene called XBP1 had a positive correlation with ATF6, BCL-2, and ERVW-1 in individuals with schizophrenia using Spearman correlation analysis. Furthermore, results from the enzyme-linked immunosorbent assay (ELISA) suggested increased serum protein levels of ATF6 and XBP1 in schizophrenic patients compared with healthy controls, exhibiting a strong correlation with ERVW-1 using median analysis and Mann-Whitney U analysis. However, serum GANAB levels were decreased in schizophrenic patients compared with controls and showed a significant negative correlation with ERVW-1, ATF6, and XBP1 in schizophrenic patients. Interestingly, in vitro experiments verified that ERVW-1 indeed increased ATF6 and XBP1 expression while decreasing GANAB expression. Additionally, the confocal microscope experiment suggested that ERVW-1 could impact the shape of the ER, leading to ER stress. GANAB was found to participate in ER stress regulated by ERVW-1. In conclusion, ERVW-1 induced ER stress by suppressing GANAB expression, thereby upregulating the expression of ATF6 and XBP1 and ultimately contributing to the development of schizophrenia.

Potential role of PRKCSH in lung cancer: bioinformatics analysis and a case study of Nano ZnO.

PRKCSH, also known as glucosidase II beta, functions as a contributor to lung tumorigenesis by regulating the cell cycle in a p53-dependent manner under severe environmental stress. However, the prognostic value and molecular mechanisms by which the level of PRKCSH is significantly increased in cancer cells are not clearly understood. Here, we first generated a biological profile of PRKCSH expression changes in cancers by analysing bioinformatic data from cancer databases. We found that higher PRKCSH expression was correlated with a poorer prognosis and greater infiltration of most immune cell types in patients with lung cancer. In particular, PRKCSH expression showed significant negative correlations with the level of STAT6 (r = -0.31, p < 0.001) in lung cancer tissues. We further found that PRKCSH deficiency promoted G2/M arrest in response to zinc oxide nanoparticle (Nano ZnO) treatment in A549 cells. With regard to the mechanism, PRKCSH deficiency may induce STAT6 translocation to the nucleus to activate p53 expression through binding to the p53 promoter region from -365 bp to +126 bp. Eventually, activated p53 contributed to Nano-ZnO-induced G2/M arrest in lung cancer cells. Taken together, our data provide new insights into immunotherapy target choices and the prognostic value of PRKCSH. Since the G2/M cell cycle checkpoint is crucial for lung cancer prognosis, targeting PRKCSH expression to suppress the activation of the STAT6/p53 pathway is a potential therapeutic strategy for managing lung cancer.

Regulation and role of glycophagy in skeletal muscle energy metabolism.

Glycophagy is the autophagic degradation of glycogen via the lysosomal enzyme GAA/alpha-acid glucosidase. Glycophagy is considered a housekeeping process to degrade poorly branched glycogen particles, but the regulation and role of glycophagy in skeletal muscle metabolism remains enigmatic. Herein, prior muscle contraction promoted glycogen supercompensation 24 and 48 h post contraction, an effect associated with reduced glycophagy. Moreover, NOTCH or cAMP signaling promoted glycophagy, whereas acute glycophagy deficiency rewired cell metabolism by reducing glycolysis and enhancing AMPK and PPAR signaling and fatty acid and glutamine metabolism. These metabolic adaptations were associated with reduced inflammation and triglyceride content but enhanced phosphoinositide 3-kinase (PI3K)-AKT/protein kinase B signaling and insulin action, the latter of which was abolished by exogenous oxidative stress. Collectively, these data suggest glycophagy is dynamically regulated, while the function of glycophagy can be extended beyond a housekeeping process to having an additional role in regulating energy metabolism and insulin action.Abbreviations: AMPK, AMP-activated protein kinase; ASM, acid soluble metabolites; cAMP, cyclic adenosine monophosphate; EPS, electrical pulse stimulation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; GAA, glucosidase, alpha, acid; mTOR, mechanistic target of rapamycin kinase; NAD, nicotinamide adenine dinucleotide; PARP, poly (ADP-ribose) polymerase family; PI3K, phosphoinositide 3-kinase; PPAR, peroxisome proliferator activated receptor ; PYGM, muscle glycogen phosphorylase; STBD1, starch binding domain 1; TFEB, transcription factor EB.

Biological activity, phytochemical parameters, and potential bioaccessibility of wheat bread enriched with powder and microcapsules made from Saskatoon berry.

The aim of the present study was to determine the impact of Saskatoon powder addition on phytochemical parameters, biological activity, and nutritional value of wheat bread. Supplementation increased phenolics content up to 72% in the bread with 6% powder content. This increase was reflected in the improved antioxidative properties of breads, especially after their supplementation with the microencapsulated additives (an increase by 93% in the bread enriched with 6% of powder covered with maltodextrin). The in vitro digestion released the antioxidative compounds, leading to higher bioaccessibility of the breads enriched with the microencapsulated powders. The highest inhibition of activities of cyclooxygenase 1 and -2, as well as amylase and glucosidase was recorded for the breads enriched with the additive microencapsulated with maltodextrin and inulin. Thus, Saskatoon berry powders, especially the microencapsulated ones, may be used as functional components in designing innovative bakery products.

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

Comparative analysis of isoflavone aglycones using microwave-assisted acid hydrolysis from soybean organs at different growth times and screening for their digestive enzyme inhibition and antioxidant properties.

The objectives of this research were to demonstrate the changes in isoflavone-aglycones, total phenolics, and biological properties (digestive enzyme inhibition; antioxidant) from six organs including leaves, leafstalks, roots, stems, seeds, and pods at different growth times of soybean plant. Three isoflavone-aglycones in microwave-assisted acid hydrolysis extracts were elucidated using UHPLC-ESI-Q-TOF-MS/MS and their contents exhibited remarkable differences in leaves (245.93-2239.33 µg/g), roots (854.96-4425.34 µg/g), and seeds (ND-2339.62 µg/g). Specifically, the collected samples on 15-Oct (leaves: 2239.33; seeds: 2339.62 µg/g) and 31-Aug (roots: 4425.34 µg/g) showed the highest isoflavone-aglycones, and daidzein was observed the most abundant component, comprising approximately 70%. Moreover, the inhibitions against α-glucosidase and α-amylase displayed the predominant effects in roots (89;91%) and leaves (81;85%) of samples on 31-Aug and 15-Oct at 300 µg/ml. The antioxidant activities on ABTS, DPPH, and hydroxyl radicals increased considerably with the increases of growth times in leaves and seeds, especially, ABTS showed the highest scavenging abilities: leaves (15-Oct;83%) > roots (31-Aug;75%) > seeds (15-Oct;68%). Therefore, our results suggest that soybean leaves, roots and seeds may be considered as excellent natural sources for nutraceuticals.

New insights into the chemical profiling, cytotoxicity and bioactivity of four Bunium species.

Bunium species have been reported to be used both as food and in traditional medicines. The scientific community has attempted to probe into the pharmacological and chemical profiles of this genus. Nonetheless, many species have not been investigated fully to date. In this study, we determined the phenolic components, antimicrobial, antioxidant, and enzyme inhibitory activities of aerial parts of four Bunium species (B. sayai, B. pinnatifolium, B. brachyactis and B. macrocarpum). Results showed that B. microcarpum and B. pinnatifolium were strong antioxidants as evidenced in the DPPH, ABTS, CUPRAC, and FRAP assays. B. brachyactis was the most effective metal chelator, and displayed high enzyme inhibition against cholinesterase, tyrosinase, amylase, glucosidase, and lipase. The four species showed varied antimicrobial activity against each microorganism. Overall, they showed high activity against P. mirabilis and E. coli (MIC and MBC <1 mg mL-1). B. brachyactis was more effective against Aspergillus versicolor compared to the standard drug ketoconazole. B. brachyactis was also more effective than both ketoconazole and bifonazole against Trichoderma viride. B. sayai was more effective than ketoconazole in inhibiting A. fumigatus. B. sayai was most non-toxic to HEK 293 (cellular viability = 117%) and HepG2 (cellular viability = 104%). The highest level of TPC was observed in B. pinnatifolium (35.94 mg GAE g-1) while B. microcarpum possessed the highest TFC (39.21 mg RE g-1). Seventy four compounds were detected in B. microcarpum, 70 in B. brachyactis, 66 in B. sayai, and 51 in B. pinnatifolium. Quinic acid, chlorogenic acid, pantothenic acid, esculin, isoquercitrin, rutin, apigenin, and scopoletin were present in all the four species. This study showed that the four Bunium species are good sources of biologically active compounds with pharmaceutical and nutraceutical potential.

PRKCSH contributes to tumorigenesis by selective boosting of IRE1 signaling pathway.

Unfolded protein response (UPR) is an adaptive mechanism that aims at restoring ER homeostasis under severe environmental stress. Malignant cells are resistant to environmental stress, which is largely due to an activated UPR. However, the molecular mechanisms by which different UPR branches are selectively controlled in tumor cells are not clearly understood. Here, we provide evidence that PRKCSH, previously known as glucosidase II beta subunit, functions as a regulator for selective activation of the IRE1α branch of UPR. PRKCSH boosts ER stress-mediated autophosphorylation and oligomerization of IRE1α through mutual interaction. PRKCSH contributes to the induction of tumor-promoting factors and to tumor resistance to ER stress. Increased levels of PRKCSH in various tumor tissues are positively correlated with the expression of XBP1-target genes. Taken together, our data provide a molecular rationale for selective activation of the IRE1α branch in tumors and adaptation of tumor cells to severe environmental stress.

Adipocyte abundances of CES1, CRYAB, ENO1 and GANAB are modified in-vitro by glucose restriction and are associated with cellular remodelling during weight regain.

Long-term weight loss maintenance is a problem of overweight and obesity. Changes of gene expression during weight loss (WL) by calorie restriction (CR) are linked to the risk of weight regain (WR). However, detailed information on genes/proteins involved in the mechanism is still lacking. Therefore, we developed an in-vitro model system for glucose restriction (GR) and refeeding (RF) to uncover proteome differences between GR with RF vs normal feeding, of which we explored the relation with WR after WL. Human Simpson-Golabi-Behmel Syndrome cells were subjected to changing levels of glucose to mimic the condition of CR and RF. Proteome profiling was performed by liquid chromatography tandem mass spectrometry. This in-vitro model revealed 44 proteins differentially expressed after GR and RF versus feeding including proteins of the focal adhesions. Four proteins showed a persistent up- or down-regulation: liver carboxylesterase (CES1), mitochondrial superoxide dismutase [Mn] (SOD2), alpha-crystallin B-chain (CRYAB), alpha-enolase (ENO1). In-vivo weight loss-induced RNA expression changes linked CES1, CRYAB and ENO1 to WR. Moreover, of these 44 proteins, CES1 and glucosidase II alpha subunit (GANAB) during follow up correlated with WR. Correlation clustering of in-vivo protein expression data indicated an interaction of these proteins with structural components of the focal adhesions and cytoplasmic filaments in the adipocytes.

Enzymatic Synthesis of ß-Glucosylglycerol and Its Unnatural Glycosides Via ß-Glycosidase and Amylosucrase.

ß-Glucosylglycerol (ß-GG) and their derivatives have potential applications in food, cosmetics and the healthcare industry, including antitumor medications. In this study, ß-GG and its unnatural glycosides were synthesized through the transglycosylation of two enzymes, Sulfolobus shibatae ß-glycosidase (SSG) and Deinococcus geothermalis amylosucrase (DGAS). SSG catalyzed a transglycosylation reaction with glycerol as an acceptor and cellobiose as a donor to produce 56% of ß-GGs [ß-D-glucopyranosyl-(1→1/3)-D-glycerol and ß-D-glucopyranosyl- (1→2)-D-glycerol]. In the second transglycosylation reaction, ß-D-glucopyranosyl-(1 → 1/3)-Dglycerol was used as acceptor molecules of the DGAS reaction. As a result, 61% of α-Dglucopyranosyl-( 1→4)-ß-D-glucopyranosyl-(1→1/3)-D-glycerol and 28% of α-D-maltopyranosyl- (1→4)-ß-D-glucopyranosyl-(1→1/3)-D-glycerol were synthesized as unnatural glucosylglycerols. In conclusion, the combined enzymatic synthesis of the unnatural glycosides of ß-GG was established. The synthesis of these unnatural glycosides may provide an opportunity to discover new applications in the biotechnological industry.

Dual targeting of PTP1B and glucosidases with new bifunctional iminosugar inhibitors to address type 2 diabetes.

The diffusion of type 2 diabetes (T2D) throughout the world represents one of the most important health problems of this century. Patients suffering from this disease can currently be treated with numerous oral anti-hyperglycaemic drugs, but none is capable of reproducing the physiological action of insulin and, in several cases, they induce severe side effects. Developing new anti-diabetic drugs remains one of the most urgent challenges of the pharmaceutical industry. Multi-target drugs could offer new therapeutic opportunities for the treatment of T2D, and the reported data on type 2 diabetic mice models indicate that these drugs could be more effective and have fewer side effects than mono-target drugs. α-Glucosidases and Protein Tyrosine Phosphatase 1B (PTP1B) are considered important targets for the treatment of T2D: the first digest oligo- and disaccharides in the gut, while the latter regulates the insulin-signaling pathway. With the aim of generating new drugs able to target both enzymes, we synthesized a series of bifunctional compounds bearing both a nitro aromatic group and an iminosugar moiety. The results of tests carried out both in vitro and in a cell-based model, show that these bifunctional compounds maintain activity on both target enzymes and, more importantly, show a good insulin-mimetic activity, increasing phosphorylation levels of Akt in the absence of insulin stimulation. These compounds could be used to develop a new generation of anti-hyperglycemic drugs useful for the treatment of patients affected by T2D.

Odd skipped-related 1 (Osr1) identifies muscle-interstitial fibro-adipogenic progenitors (FAPs) activated by acute injury.

Fibro-adipogenic progenitors (FAPs) are resident mesenchymal progenitors in adult skeletal muscle that support muscle repair, but also give rise to fibrous and adipose infiltration in response to disease and chronic injury. FAPs are identified using cell surface markers that do not distinguish between quiescent FAPs and FAPs actively engaged in the regenerative process. We have shown previously that FAPs are derived from cells that express the transcription factor Osr1 during development. Here we show that adult FAPs express Osr1 at low levels and frequency, however upon acute injury FAPs reactivate Osr1 expression in the injured tissue. Osr1+ FAPs are enriched in proliferating and apoptotic cells demonstrating that Osr1 identifies activated FAPs. In vivo genetic lineage tracing shows that Osr1+ activated FAPs return to the resident FAP pool after regeneration as well as contribute to adipocytes after glycerol-induced fatty degeneration. In conclusion, reporter LacZ or eGFP-CreERt2 expression from the endogenous Osr1 locus serves as marker for FACS isolation and tamoxifen-induced manipulation of activated FAPs.

Multiplex Fluorescent, Activity-Based Protein Profiling Identifies Active α-Glycosidases and Other Hydrolases in Plants.

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-glycosidase inhibitors in a broad range of plant species. To demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active α-glycosidases and other hydrolases can be analyzed through ABPP in model and nonmodel plants.

Geissoschizine synthase controls flux in the formation of monoterpenoid indole alkaloids in a Catharanthus roseus mutant.

MAIN CONCLUSION: A Catharanthus roseus mutant accumulates high levels of ajmalicine at the expense of catharanthine and vindoline. The altered chemistry depends on increased expression and biochemical activities of strictosidine ß-glucosidase and ajmalicine synthase activities and reduced expression and biochemical activity of geissoschizine synthase. The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is a valuable source for several monoterpenoid indole alkaloids (MIAs), such as the powerful antihypertensive drug ajmalicine and the antineoplastic agents, vinblastine and vincristine. While biosynthesis of the common MIA precursor strictosidine and its reactive aglycones has been elucidated, the branch point steps leading to the formation of different classes of MIAs remain poorly characterized. Screening of 3600 ethyl methyl sulfonate mutagenized C. roseus plants using a simple thin-layer chromatography screen yielded a mutant (M2-0754) accumulating high levels of ajmalicine together with significantly lower levels of catharanthine and vindoline. Comparative bioinformatic analyses, virus-induced gene silencing, and biochemical characterization identified geissoschizine synthase, the gateway enzyme that controls flux for the formation of iboga and aspidosperma MIAs. The reduction of geissoschizine synthase transcripts in this high ajmalicine mutant, together with increased transcripts and enzyme activities of strictosidine ß-glucosidase and of heteroyohimbine synthase, explains the preferential formation of ajmalicine in the mutant instead of catharanthine and vindoline that accumulates in the wild-type parent. Reciprocal crosses established that that the high ajmalicine phenotype is inherited as a Mendelian recessive trait.

Effect of in vitro simulated gastrointestinal digestion on polyphenol and polysaccharide content and their biological activities among 22 fruit juices.

Polyphenols and polysaccharides, as natural bioactive compounds from common fresh fruits, are concerned in reducing risk of developing obesity and diabetes for human in recent years. The content of polyphenol and polysaccharide, their bioactivities among 22 fruit juices were investigated before and after in vitro gastrointestinal digestion in present study. After digestion, contents of polyphenol, polysaccharide and their antioxidant activity, the inhibitory activity of α-amylase and α-glucosidase significantly increased. Punica granatum Linn and Actinidia globosa C. F. Liang displayed maximal increment up to 2, 0.25 and 1.6 fold in contents of polyphenols and polysaccharides, and the inhibitory activity of α-amylase, respectively. The correlation coefficient between contents and inhibitory activity of α-amylase increased in range of 0.002 to 0.485. Lycopersicon esculentum Mill and Pyrus bretschneideri Rehd exhibited maximum increase in the inhibitory activity of α-glucosidase with lowest contents of polyphenols and polysaccharides. The results indicated that polyphenols and polysaccharides digested synergistically contributing to the inhibitory α-amylase activity, and other responsibly bioactive ingredients for inhibitory α-glucosidase activity would be worthy discussed future. The findings above highlighted some potential application of common fruit juices in controlling hyperglycemia and obesity.

Liver cyst gene knockout in cholangiocytes inhibits cilium formation and Wnt signaling.

Mutations in the PRKCSH, SEC63 and LRP5 genes cause autosomal dominant polycystic liver disease (ADPLD). The proteins products of PRKCSH (alias GIIB) and SEC63 function in protein quality control and processing in the endoplasmic reticulum (ER), while LRP5 is implicated in Wnt/ß-catenin signaling. To identify common denominators in the PLD pathogenesis, we mapped the PLD interactome by affinity proteomics, employing both HEK293T cells and H69 cholangiocytes. Identification of known complex members, such as glucosidase IIA (GIIA) for PRKCSH, and SEC61A1 and SEC61B for SEC63, confirmed the specificity of the analysis. GANAB, encoding GIIA, was very recently identified as an ADPLD gene. The presence of GIIA in the LRP5 complex pinpoints a potential functional connection with PRKCSH. Interestingly, all three PLD-associated protein complexes included filamin A (FLNA), a multifunctional protein described to play a role in ciliogenesis as well as canonical Wnt signalling. As ciliary dysfunction may also contribute to hereditary liver cyst formation, we evaluated the requirement of PRKCSH and SEC63 for ciliogenesis and Wnt signaling. By CRISPR/Cas9 induced knockdown of both ADPLD genes in HEK293T cells and H69 cholangiocytes, we identified that their depletion results in defective ciliogenesis. However, only H69 knockouts displayed reduced Wnt3a activation. Our results suggest that loss of PRKCSH and SEC63 leads to general defects in ciliogenesis, while quenching of the Wnt signaling cascade is cholangiocyte-restricted. Interactions of all three PLD-associated protein complexes with FLNA may mark a common link between the ADPLD proteins and the cystogenic processes driving this disease.

Glucosidase II beta subunit (GluIIß) plays a role in autophagy and apoptosis regulation in lung carcinoma cells in a p53-dependent manner.

PURPOSE: Glucosidase II plays a major role in regulating the post-translational modification of N-linked glycoproteins. Previously, we found that the beta subunit of glucosidase II (GluIIß) levels are significantly increased in lung carcinoma tissues, indicating a potential role in lung tumorigenesis. Here, we investigated the role of GluIIß in the regulation of autophagy and apoptosis in lung carcinoma- and immortalized human bronchial epithelial-derived cells. METHODS: A selective glucosidase II inhibitor, bromoconduritol, was used to inhibit GluII enzyme activity and a siRNA-based technology was used to suppress the expression of the GluIIß encoding gene PRKCSH in lung carcinoma cells differing in p53 status. Cell viability was assessed using a MTT assay, cell cycle progression was assessed using flow cytometry, autophagy was assessed using Western blotting and apoptosis was assessed using an annexin V-FITC/PI double labeling method. RESULTS: We found that GluIIß inhibition resulted in the induction of autophagy in all cell lines tested, but apoptosis in only wild-type p53 cells. We also found that GluIIß inhibition dose-dependently decreased activation of the EGFR/RTK and PI3K/AKT signaling pathways. Although the apoptosis inducing effect of GluIIß inhibition appeared to be p53-dependent, we found that a combined treatment with lysosomal inhibitors to block autophagy enhanced the apoptotic effect of GluIIß inhibition in both wild-type p53 and p53-null cells. CONCLUSIONS: Our data indicate that GluIIß inhibition results in autophagy and apoptosis in lung carcinoma-derived cells, supporting the hypothesis that this enzyme may play a role in blocking these two tumor suppressive processes. Since blocking autophagy by lysosomal inhibitors enhanced the apoptosis-inducing effect of bromoconduritol, independent of p53 status, their combined use may hold promise for the treatment of cancer, particularly lung cancer.

Structural basis of the substrate specificity and instability in solution of a glycosidase from Lactobacillus plantarum.

Statistics from structural genomics initiatives reveal that around 50-55% of the expressed, non-membrane proteins cannot be purified and therefore structurally characterized due to solubility problems, which emphasized protein solubility as one of the most serious concerns in structural biology projects. Lactobacillus plantarum CECT 748T produces an aggregation-prone glycosidase (LpBgl) that we crystallized previously. However, this result could not be reproduced due to protein instability and therefore further high-resolution structural analyses of LpBgl were impeded. The obtained crystals of LpBgl diffracted up to 2.48Å resolution and permitted to solve the structure of the enzyme. Analysis of the active site revealed a pocket for phosphate-binding with an uncommon architecture, where a phosphate molecule is tightly bound suggesting the recognition of 6-phosphoryl sugars. In agreement with this observation, we showed that LpBgl exhibited 6-phospho-ß-glucosidase activity. Combination of structural and mass spectrometry results revealed the formation of dimethyl arsenic adducts on the solvent exposed cysteine residues Cys211 and Cys292. Remarkably, the double mutant Cys211Ser/Cys292Ser resulted stable in solution at high concentrations indicating that the marginal solubility of LpBgl can be ascribed specifically to these two cysteine residues. The 2.30Å crystal structure of this double mutant showed no disorder around the newly incorporated serine residues and also loop rearrangements within the phosphate-binding site. Notably, LpBgl could be prepared at high yield by proteolytic digestion of the fusion protein LSLt-LpBgl, which raises important questions about potential hysteretic processes upon its initial production as an enzyme fused to a solubility enhancer.

Isolated polycystic liver disease genes define effectors of polycystin-1 function.

Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALG8, GANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases.