Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5–P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds.     

Differential roles of RIPK1 and RIPK3 in TNF-induced necroptosis and chemotherapeutic agent-induced cell death

Apoptosis is a key mechanism for metazoans to eliminate unwanted cells. Resistance to apoptosis is a hallmark of many cancer cells and a major roadblock to traditional chemotherapy. Recent evidence indicates that inhibition of caspase-dependent apoptosis sensitizes many cancer cells to a form of non-apoptotic cell death termed necroptosis. This has led to widespread interest in exploring necroptosis as an alternative strategy for anti-cancer therapy. Here we show that in human colon cancer tissues, the expression of the essential necroptosis adaptors receptor interacting protein kinase (RIPK)1 and RIPK3 is significantly decreased compared with adjacent normal colon tissues. The expression of RIPK1 and RIPK3 was suppressed by hypoxia, but not by epigenetic DNA modification. To explore the role of necroptosis in chemotherapy-induced cell death, we used inhibitors of RIPK1 or RIPK3 kinase activity, and modulated their expression in colon cancer cell lines using short hairpin RNAs. We found that RIPK1 and RIPK3 were largely dispensable for classical chemotherapy-induced cell death. Caspase inhibitor and/or second mitochondria-derived activator of caspase mimetic, which sensitize cells to RIPK1- and RIPK3-dependent necroptosis downstream of tumor necrosis factor receptor-like death receptors, also did not alter the response of cancer cells to chemotherapeutic agents. In contrast to the RIPKs, we found that cathepsins are partially responsible for doxorubicin or etoposide-induced cell death. Taken together, these results indicate that traditional chemotherapeutic agents are not efficient inducers of necroptosis and that more potent pathway-specific drugs are required to fully harness the power of necroptosis in anti-cancer therapy.Cell death by apoptosis is a natural barrier to cancer development, as it limits uncontrolled proliferation driven by oncogenes.¹ Chemotherapeutic agents that target apoptosis have been successful in anti-cancer therapy. However, cancer cells, especially cancer stem cells, often evolve multiple mechanisms to circumvent growth suppression by apoptosis.² This resistance to apoptosis is a major challenge for many chemotherapeutic agents. Targeting other non-apoptotic cell death pathways is an attractive therapeutic alternative.A growing number of recent studies show that there are distinct genetic programmed cell death modes other than apoptosis.³ Necroptosis is mediated by receptor interacting protein kinase 3 (RIPK3).⁴ In the presence of caspase inhibition and cellular inhibitor of apoptosis proteins (cIAPs) depletion, tumor necrosis factor (TNF) receptor 1 triggers a signaling reaction that culminates in binding of RIPK3 with its upstream activator RIPK1 through the RIP homotypic interaction motif (RHIM).⁴ RIPK1 and RIPK3 phosphorylation stabilizes this complex and promotes its conversion to an amyloid-like filamentous structure termed the necrosome.⁵ Once activated, RIPK3 recruits its substrate mixed lineage kinase domain-like (MLKL).⁶ Phosphorylated MLKL forms oligomers that translocate to intracellular membranes and the plasma membrane, which eventually leads to membrane rupture.^{7, 8, 9, 10}In addition to phosphorylation, RIPK1 and RIPK3 are also tightly regulated by ubiquitination, a process mediated by the E3 ligases cIAP1, cIAP2, and the linear ubiquitin chain assembly complex.¹¹ The ubiquitin chains on RIPK1 act as a scaffold to activate nuclear factor-κB (NF-κB) and mitogen-activated protein kinase pathways and inhibit formation of the necrosome. As such, depletion of cIAP1/2 by second mitochondria-derived activator of caspase (Smac) mimetics or removal of the ubiquitin chains by the de-ubiquitinating enzyme cylindromatosis (CYLD) promotes necroptosis.^{12, 13, 14, 15} In addition, RIPK1 and RIPK3 are cleaved and inactivated by caspase 8.^{16, 17, 18} Mice deficient for caspase 8 or FADD, an essential adaptor protein of caspase 8, suffer from embryonic lethality due to extensive RIPK1- or RIPK3-dependent necroptosis.^{19, 20, 21} Hence, caspase inhibition and IAP depletion are key priming signals for necroptosis.The physiological functions of RIPK1 and RIPK3 have been extensively investigated in infectious and sterile inflammatory diseases.^{4, 22} By contrast, their roles in cancer cells'' response to chemotherapeutics are poorly understood. Here we show that RIPK1 and RIPK3 expression is significantly decreased in human colon cancer tissues, suggesting that suppression of RIPK1 or RIPK3 expression is advantageous for cancer growth. However, the loss of RIPK1 and RIPK3 expression in colon cancer was not due to epigenetic DNA modification. Interestingly, RIPK1 and RIPK3 expression in colon cancer cells is reduced by hypoxia, a hallmark of solid tumor. We found that chemotherapeutic agents did not effectively elicit RIPK1/RIPK3-dependent necroptosis in colon cancer cells. Moreover, caspase inhibition and Smac mimetics, which are potent sensitizers for necroptosis, also did not enhance chemotherapeutic agent-induced cell death. These results show that traditional chemotherapeutic agents are not strong inducers of classical necroptosis in colon cancers and suggest that development of pathway-specific drugs is needed to harness the power of necroptosis in anti-cancer therapy.     

Hallmarks of atopic skin mimicked in vitro by means of a skin disease model based on FLG knock-down

Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knock-down model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.     

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.

Results

For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.

Conclusions

The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.     

Melatonin suppresses AOM/DSS-induced large bowel oncogenesis in rats

The inhibitory effects of exogenous melatonin (MEL) on colon oncogenesis were investigated using an azoxymethane (AOM)/dextran sodium sulfate (DSS) rat model. Male F344 rats initiated with a single intraperitoneal injection of AOM (20 mg/kg bw) were promoted by 1% (w/v) DSS in drinking water for 7 days. They were then given 0.4, 2 or 10 ppm MEL in drinking water for 17 weeks. At week 20, the development of colonic adenocarcinoma was significantly inhibited by the administration with MEL dose-dependently. MEL exposure modulated the mitotic and apoptotic indices in the colonic adenocarcinomas that developed and lowered the immunohistochemical expression of nuclear factor kappa B, tumor necrosis factor α, interleukin-1β and STAT3 in the epithelial malignancies. These results may indicate the beneficial effects of MEL on colitis-related colon carcinogenesis and a potential application for inhibiting colorectal cancer development in the inflamed colon.     

Cyto- and genotoxic potential of beta-carotene and cleavage products under oxidative stress

Free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria, a finding which could provide an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators for the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo-8'-beta-carotenal (apo-8') and beta-carotene in the primary rat hepatocyte assay in the presence and absence of oxidative stress provided by hypoxia/reoxygenation (Hy/re). The endpoints tested were: the mitotic indices, the percentages of necrotic and apoptotic cells, micronucleated cells (MN), chromosomal aberrations (CA) and sister chromatid exchanges (SCE). The results obtained indicate a genotoxic potential of both CP and apo-8' already in the concentration range of 100 nM and 1 microM, i.e. at physiologically relevant levels of beta-carotene and beta-carotene breakdown products. In contrast, no significant cytotoxic effects of these substances were observed, nor did beta-carotene induce significant cytotoxic or genotoxic effects at concentrations ranging from 0.01 up to 10 microM. However, when beta-carotene is supplemented during oxidative stress induced by hypoxia/reoxygenation, a dose-dependent increase of CP is observed accompanied by increasing genotoxicity. Furthermore, when beta-carotene cleavage products were supplied during oxidative stress significant additional increases of genotoxic effects were observed, the additional increases indicating an additive effect of both exposures. Summarizing, these results provide strong evidence that beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-carotene-Cancer prevention (ATBC) study and the beta-CArotene and RETinol Efficacy (CARET) Trial.     

Allelic heterogeneity in the COH1 gene explains clinical variability in Cohen syndrome

Cohen syndrome is a rare autosomal recessive disorder with a variable clinical picture mainly characterized by developmental delay, mental retardation, microcephaly, typical facial dysmorphism, progressive pigmentary retinopathy, severe myopia, and intermittent neutropenia. A Cohen syndrome locus was mapped to chromosome 8q22 in Finnish patients, and, recently, mutations in the gene COH1 were reported in patients with Cohen syndrome from Finland and other parts of northern and western Europe. Here, we describe clinical and molecular findings in 20 patients with Cohen syndrome from 12 families, originating from Brazil, Germany, Lebanon, Oman, Poland, and Turkey. All patients were homozygous or compound heterozygous for mutations in COH1. We identified a total of 17 novel mutations, mostly resulting in premature termination codons. The clinical presentation was highly variable. Developmental delay of varying degree, early-onset myopia, joint laxity, and facial dysmorphism were the only features present in all patients; however, retinopathy at school age, microcephaly, and neutropenia are not requisite symptoms of Cohen syndrome. The identification of novel mutations in COH1 in an ethnically diverse group of patients demonstrates extensive allelic heterogeneity and explains the intriguing clinical variability in Cohen syndrome.     

Territorial defense against various food competitors in the Tanganyikan benthophagous cichlid <Emphasis Type="Italic">Neolamprologus tetracanthus</Emphasis>

Territorial defense of nonbreeding female Neolamprologus tetracanthus, a shrimp-eating Tanganyikan cichlid, was investigated. Females defended territories (=home ranges, ca. 1m across) against a variety of intruding fishes. Conspecific females were usually attacked outside the territories, heterospecific benthivores (shrimp eaters) and omnivores near the border of the territories, and piscivores, algae and detritus feeders, and herbivores inside the territories. Females used some parts of the sandy substrate in the territories for foraging (foraging areas). Territorial defense prevented most of the conspecific females and benthivores from intruding into the foraging areas. In omnivores, piscivores, and algae and detritus feeders, about half the intruders were repelled from the foraging areas, although herbivores were infrequently repelled in the areas. Soon after removal of the resident females, many food competitors invaded the foraging areas and eagerly devoured prey, suggesting that the territories are maintained for food resource protection from these competitors. Females are likely to discriminate intruding fishes and change their territorial defense primarily on the basis of the degree of dietary overlap, resulting in monofunctional serial territories.     

Avidin related protein 2 shows unique structural and functional features among the avidin protein family

Background  

The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.