Repositioning of Verrucosidin,a Purported Inhibitor of Chaperone Protein GRP78, as an Inhibitor of Mitochondrial Electron Transport Chain Complex I

Verrucosidin (VCD) belongs to a group of fungal metabolites that were identified in screening programs to detect molecules that preferentially kill cancer cells under glucose-deprived conditions. Its mode of action was proposed to involve inhibition of increased GRP78 (glucose regulated protein 78) expression during hypoglycemia. Because GRP78 plays an important role in tumorigenesis, inhibitors such as VCD might harbor cancer therapeutic potential. We therefore sought to characterize VCD’s anticancer activity in vitro. Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with VCD under different conditions known to trigger increased expression of GRP78, and a variety of cellular processes were analyzed. We show that VCD was highly cytotoxic only under hypoglycemic conditions, but not in the presence of normal glucose levels, and VCD blocked GRP78 expression only when glycolysis was impaired (due to hypoglycemia or the presence of the glycolysis inhibitor 2-deoxyglucose), but not when GRP78 was induced by other means (hypoxia, thapsigargin, tunicamycin). However, VCD’s strictly hypoglycemia-specific toxicity was not due to the inhibition of GRP78. Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain. As a result, cellular ATP levels were quickly depleted under hypoglycemic conditions, and common cellular functions, including general protein synthesis, deteriorated and resulted in cell death. Altogether, our study identifies mitochondria as the primary target of VCD. The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin) might act in a similar GRP78-independent fashion will be discussed.     

Cyclic strain stimulates proliferative capacity, alpha2 and alpha5 integrin, gene marker expression by human articular chondrocytes propagated on flexible silicone membranes

Chondrocytes comprise less than 10% of cartilage tissue but are responsible for sensing and responding to mechanical stimuli imposed on the joint. However, the effect of mechanical signals at the cellular level is not yet fully defined. The purpose of this study was to test the hypothesis that mechanical stimulation in the form of cyclic strain modulates proliferative capacity and integrin expression of chondrocytes from osteoarthritic knee joints. Chondrocytes isolated from articular cartilage during total knee arthroplasty were propagated on flexible silicone membranes. The cells were subjected to cyclic strain for 24 h using a computer-controlled vacuum device, with replicate samples maintained under static conditions. Our results demonstrated increase in proliferative capacity of the cells subjected to cyclic strain compared with cells maintained under static conditions. The flexed cells also exhibited upregulation of the chondrocytic gene markers type II collagen and aggrecan. In addition, cyclic strain resulted in increased expression of the alpha2 and alpha5 integrin subunits, as well as an increased expression of vimentin. There was also intracellular reconfiguration of the enzyme protein kinase C. Our findings suggest that these molecules may play a role in the signal transduction pathway, eliciting cellular response to mechanical stimulation.     

Ferritin and ferritin isoforms II: protection against uncontrolled cellular proliferation, oxidative damage and inflammatory processes

Ferritin is a major iron storage protein involved in the regulation of iron availability. Each ferritin molecule comprises 24 subunits. Various combinations of H-subunits and L-subunits make up the 24-subunit protein structure and these ferritin isoforms differ in their H-subunit to L-subunit ratio, as well as in their metabolic properties. Ferritin is an acute-phase protein and its expression is up-regulated in conditions such as uncontrolled cellular proliferation, in any condition marked by excessive production of toxic oxygen radicals, and by infectious and inflammatory processes. Under such conditions ferritin up-regulation is predominantly stimulated by increased reactive oxygen radical production and by cytokines. The major function of ferritin in these conditions is to reduce the bio-availability of iron in order to stem uncontrolled cellular proliferation and excessive production of reactive oxygen radicals. Ferritin is not, however, indiscriminately up-regulated in these conditions as a marked shift towards a predominance in H-subunit rich ferritins occurs. Preliminary indications are that, while the L-subunit primarily fulfils the conventional iron storage role, the H-subunit functions primarily as rapid regulator of iron availability, and perhaps indirectly as regulator of other cellular processes. It is suggested that the optimum differential expression of the two subunits differ for different cells and under different conditions and that the expression of appropriate isoferritins offers protection against uncontrolled cellular proliferation, oxidative stress and against side effects of infectious and inflammatory conditions.     

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.     

Cellular expression of murine Ym1 and Ym2, chitinase family proteins,as revealed by in situ hybridization and immunohistochemistry

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.     

High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system

N-substituted formamide deformylase (NfdA) from Arthrobacter pascens F164 is a novel deformylase involved in the metabolism of isonitriles. The enzyme catalyzes the deformylation of an N-substituted formamide, which is produced from the corresponding isonitrile, to yield the corresponding amine and formate. The nfdA gene from A. pascens F164 was cloned into different types of expression vectors for Escherichia coli and Streptomyces strains. Expression in E. coli resulted in the accumulation of an insoluble protein. However, Streptomyces strains transformed with a P(nitA)-NitR system, which we very recently developed as a regulatory gene expression system for streptomycetes, allowed the heterologous overproduction of NfdA in an active form. When Streptomyces lividans TK24 transformed with pSH19-nfdA was cultured under the optimum conditions, the NfdA activity of the cell-free extract amounted to 8.5 U/mg, which was 29-fold higher than that of A. pascens F164. The enzyme also comprised approximately 20% of the total extractable cellular protein. The recombinant enzyme was purified to homogeneity and characterized. The expression system established here will allow structural analysis and mutagenesis studies of NfdA.     

Proteomic analysis of global protein expression changes in the endothelin-1 rat model for cerebral ischemia: Rescue effect of mild hypothermia

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24 h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.     

Construction of an overproducing strain,purification, and biochemical characterization of the 6His-Eco29kI restriction endonuclease

We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter. The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein. We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay. The isolated enzymes were tested for recognition specificity and optimal reaction conditions as a function of NaCl and KCl concentrations, temperature, and pH compared with the native Eco29kI restriction endonuclease. The 6His-tagged enzyme retained the specificity of the native protein but had an altered optimum of its catalytic reaction.     

Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity.

The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2 (cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11.cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2 immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10(-6) M). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.     

Altered response to growth factors in rat epithelial liver cells overexpressing human c-Fos protein.

Human c-fos cDNA was transfected into normal rat liver epithelial (REL) cells to identify cellular modifications associated with high expression of c-Fos protein. Responses to EGF and TGF beta were examined in the different cell lines, under anchorage-dependent and -independent conditions. Sensitivity to both factors was modified in transfected cells. While parental cells in monolayer did not respond to EGF, c-fos containing cells growth was stimulated by this factor. Overexpression of c-Fos protein led to an enhanced TGF beta-induced growth inhibition under anchorage dependent conditions, and TGF beta abolished spontaneous growth in soft agar of the cell lines containing c-fos oncogene. The mechanisms underlying the increased sensitivity to TGF beta in c-fos transfected cells are still to be determined.     

Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response

Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.