Autolytic proteolysis within the function to find domain (FIIND) is required for NLRP1 inflammasome activity

Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser(1213) within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His(1186). A disease-associated single nucleotide polymorphism near His(1186) and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.     

Absence of RIPK3 predicts necroptosis resistance in malignant melanoma

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms. In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma. Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors. Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3. Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion. Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling. Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present. Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis. Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.Over the past few years, necroptosis has been established as an alternative programmed form of cell death, contrasting caspase-dependent apoptosis. It is now evident that an ordered activation of the receptor-interacting protein kinases-1 and -3 (RIPK1 and RIPK3), and their downstream substrates is mandatory for the execution of necroptosis.^{1, 2, 3} Under caspase-limited conditions, the necroptotic cell signalling machinery is regulated by RIPK1, with the impact of scaffolding function as compared with kinase function still unclear.^{1, 4, 5, 6} RIPK1 interacts with and either autophosphorylates or transphosphorylates RIPK3 (for review, see Cho et al.,¹ Zhang et al.,² He et al.,³ and Vanden Berghe et al.⁷). When RIPK1 is active, RIPK3 phosphorylation and activation occurs within the assembled Necrosome (for review, see Remijsen et al.⁸) or Ripoptosome.^{4, 9, 10} RIPK3 then phosphorylates the pseudo kinase mixed-lineage kinase domain-like protein (MLKL).¹¹ MLKL in its active form allows its oligomerization, membrane accumulation, and complex formation within cellular membranes of the mitochondria¹² and cell membranes,¹³ and finally results in necroptosis.¹⁴The RIPK1/RIPK3/MLKL signalling network acts as a sensor for genotoxic stress⁹ and also has a key role in necroptosis regulation in keratinocyte skin cancer (SCC).⁴ In these epithelial cancers, cellular inhibitors of apoptosis proteins (cIAPs) block both apoptotic and necroptotic cell death.^{4, 5} Both apoptosis and necroptosis can be increasingly initiated by intrinsic or extrinsic stimuli when IAPs are suppressed by IAP antagonist. Extrinsic apoptosis mediated by activation of death receptors (DRs) such as cluster of differentiation 95 (CD95), TRAILR1/R2 or tumour necrosis factor receptor-1 (TNFR1) through ligation of respective death ligands (DLs) such as CD95L, TNF-related apoptosis-inducing ligand (TRAIL), and TNF initiates apoptosis either by direct activation of the caspase cascade (caspase-8/caspase-3) or via the intrinsic cell death signalling machinery regulated by pro-apoptotic members of the Bcl-2 family followed by caspase-3 activation.¹⁵ Inhibition of caspase-8 within the death-inducing signalling complex or complex II, or within the Ripoptosome can trigger CD95L-mediated,⁵ TRAIL-mediated¹⁶ or TNF-induced necroptosis.^{8, 17} A role for apoptosis resistance, cancer maintenance, and progression is widely assumed (for review, see Obexer et al.¹⁸), but the pathophysiological inhibitory or propagating function of necroptosis has not formally been demonstrated in cancer.Metastatic melanoma has an overall poor prognosis but novel therapeutics have revolutionized clinical practice for different subsets of patients. The use of inhibitors of the V600E- or V600K-mutated proto-oncogene serine/threonine protein kinase B-RAF (e.g., Dabrafenib or Vemurafenib) results in suppression of Ras/Raf/mitogen-activated protein kinase pathways and translate into unfortunately transient clinical responses (for review, see Spagnolo et al.¹⁹). The high recrudescence of metastatic melanoma following the treatment with BRAF inhibitors will potentially require combination therapies that activate additional tumour-inhibitory pathways. Combinations such as BRAF inhibitors with mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitors have already yielded impressive results²⁰ and other combination therapies may further improve clinical outcome.²¹ As BRAF inhibitors target the cell death pathway at best in an indirect manner, we reasoned that necroptosis induction could represent a novel option to improve melanoma therapy. Our investigations demonstrate for the first time that loss of RIPK3 during melanoma development is critical for necroptosis protection. Reactivation of the RIPK1/RIPK3/MLKL signalling machinery by RIPK3 reconstitution allows IAP antagonist/DL-mediated necroptosis in the presence of Vemurafenib, but not Dabrafenib. Here, Dabrafenib blocks necroptosis by interference with RIPK3-mediated MLKL phosphorylation. Therefore, strategies that increase RIPK3 expression in combination with Vemurafenib, but not Dabrafenib, likely represent an attractive strategy to overcome cell death resistance in melanoma.     

On processing field and culture samples of desmids (Desmidiales,chlorophyta) for scanning electron microscopy

A procedure is outlined for preparing both field collected and laboratory grown desmid populations for scanning electron microscopy which avoids the use of the potentially hazardous chemicals glutaraldehyde and osmium tetroxide and reduces preparation time substantially. FAA is utilized both in fixation and mucilage removal, and living material can be prepared for critical point drying in as little as 90 min. Limitations of this procedure are outlined briefly and the use of SEM material in systematic studies is commented upon.     

Development of multiple strain competitive index assays for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using pIMC; a new site-specific integrative vector

Background  

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.     

Movement of adult Atlantic salmon in the Usk estuary, Wales

A total of 56 salmon was tagged in the Usk estuary using combined acoustic and radio tags. Those fish migrating within the estuary oscillated with the tide over c . 10 km, being towards the seaward end at low water and moving upstream on the flood tide. Fish migrating through the estuary moved upstream on the flood tide and stemmed displacement downstream during the ebb. These findings, together with information on the hydrodynamics of the estuary, indicate that the fish utilize tidal currents to migrate passively in their preferred direction.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner

Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD⁺) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD⁺ promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD⁺-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD⁺ does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.Nicotinamide adenine dinucleotide (NAD⁺) has been long recognized as a key intermediate in cellular metabolism. By accepting and donating electrons in reactions catalyzed by dehydrogenases, NAD⁺ has, for example, a central role in the generation of ATP, a molecule required for most energy-consuming cellular reactions. The recognition of NAD⁺ as a substrate in a number of regulatory processes has shed a new light on its role in cell physiology. Indeed, NAD⁺ represents a substrate for a wide range of enzymes including cADP-ribose synthases, poly (ADP-ribose) polymerases (PARPs) and the sirtuin family of NAD⁺-dependent deacylases (SIRTs). In marked contrast to its role in energy metabolism, the involvement of NAD⁺ in these enzymatic reactions is based on its ability to function as a donor of ADP-ribose, a reaction that, if sustained, can lead to the depletion of the intracellular NAD⁺ pool.^{1, 2, 3, 4, 5}The pro-survival role of NAD⁺ has been particularly well described in cells exposed to genotoxic/oxidative stress. In response to DNA damage, PARP1, the founding and most abundant member of the PARP family, binds to DNA strand breaks and initiates a repair response by catalyzing the post-translational modification of several nuclear proteins, including itself. This protective response is characterized by the transfer of successive units of the ADP-ribose moiety (up to 200 units) from NAD⁺ to other proteins, compromising therefore both energy production (slowing the rate of glycolysis, electron transport and ATP formation) and activity of other NAD⁺-dependent enzymes through NAD⁺ depletion.^{6, 7} Moreover, PARP1-synthesized PAR polymers can be degraded into free oligomers, known to translocate to the mitochondria where they can trigger the release of AIF from mitochondria to the nucleus.^{8, 9, 10, 11} The precise molecular steps linking PARP1 activation to this form of stress-induced cell death, termed parthanatos, have not been fully elucidated, and probably depend on the particular metabolic status of the cell examined (i.e., anerobic glycolysis in most in vitro cell lines versus oxidative metabolism of neuronal cells, see Welsby et al.¹² for review). In any instances, and independently of the fine molecular events at work, virtually all studies have identified the brisk loss of intracellular NAD⁺ as the critical step initiating this specific form of cell death. The protective role of NAD⁺ in PARP1-dependent cell death has been indeed amply documented.^{13, 14, 15, 16, 17, 18} In mammals, nicotinamide (Nam) acts as the main precursor for NAD⁺ biosynthesis and nicotinamide phosphoribosyl tranferase (NAMPT) is the rate-limiting enzyme for NAD⁺ biosynthesis from Nam.¹⁹ Nampt deficiency in mice leads to lethality and the heterozygous animals suffer from significant perturbations related to their NAD⁺ metabolism.²⁰ In keeping with the general role of NAD⁺ as a survival factor in cells exposed to genotoxic stress, genetic ablation of Nampt and/or treatment with a specific NAMPT inhibitor (FK866) sensitized cells to the toxic effects of alkylating agents.^{16, 18} Similarly, overexpression of a catalytically active recombinant NAMPT protected the NIH-3T3 cell line from the toxicity of the same DNA alkylating agents,¹⁸ further establishing a functional link between NAD⁺ biosynthesis and sensitivity to stress-induced, PARP1-dependent cell death.While analyzing the influence of NAD metabolism on survival of NIH3T3 cells exposed to genotoxic agents, we observed that overexpression of NAMPT prolonged cell survival of cells exposed to the alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), and unexpectedly, led to increased sensitivity to cell death induced by the pro-inflammatory cytokine TNF.¹⁸ TNF is a pleiotropic cytokine regulating many cellular functions and known to induce several forms of cell death, including apoptosis and the recently uncovered regulated form of necrosis termed necroptosis.^{21, 22} In contrast to apoptosis, necroptosis is largely independent of the so-called executioner caspase (such as caspase-3, 6 and 7) activity and is initiated by the formation of a signaling complex comprising the receptor-interacting serine-threonine kinase 1 (RIPK1), RIPK3 and the recently identified mixed lineage kinase domain-like protein MLKL. Although necroptosis often appears to occur when apoptosis is abortive (such as in situations of caspase inhibition), the cellular factors regulating the choice between these two forms of regulated cell death have not been fully uncovered. Using a model cell line engineered to respond to both apoptosis and necroptosis, we demonstrate herein that intracellular NAD⁺ represents a critical factor in promoting cell death by necroptosis. In keeping with the well-described role of sirtuins as intracellular NAD⁺ sensors, we also demonstrate that sirtuins, and in particular SIRT2 and SIRT5, are required for adequate completion of the necroptotic program in response to TNF. Accordingly, a pan-sirtuin inhibitor was found to attenuate organ damage induced by transient ischemia. Thus, intracellular NAD⁺, rather than acting as a general cell survival factor, appears to promote cell necroptosis in a sirtuin-dependent manner, a finding that may suggest novel therapeutic approaches to attenuate in vivo necrotic insults in several pathological settings.     

The human cytochrome P450 CYP3 locus: assignment to chromosome 7q22-qter

Summary DNA haplotypes (HT) and frameworks (FW) linked to the -globin locus were determined by restriction fragment analysis using eight restriction enzymes on chromosomes bearing the HbA gene (HBB^*A) or the HbE gene (HBB^*E) in the So, an Austro-Asiatic population of northeast Thailand with an HBB^*E frequency near 0.5. All HBB^*E genes were present with FW2, and only two haplotypes were observed (25 HT 27-2,-+-++++-; 10 HT 41-2, +----++-). In a control group from the general population of Northeast Thailand the HT distribution was more diverse, and 2 of 20 HBB^*E genes were present in FW 3. High frequencies of HBB^*E in FW 3 in Southeast Asia are apparently limited to the Khmer population of Cambodia. There were no differences in the hematologic parameters in subjects homozygous for HBB^*E/FW2 or HBB^*E/FW3.     

Altered DNA synthesis in a mutant of Salmonella typhimurium that channels bacteriophage P22 toward lysogeny.

Pox-1, a mutant of Salmonella typhimurium, strongly channels P22 toward lysogeny. Viral DNA synthesis in this slow-growing mutant is delayed to a greater extent than viral protein synthesis. The relative enhancement of c2 repressor synthesis results in much higher repressor/DNA synthesis ratios in Pox-1 than in wild-type cells. This probably accounts for the high frequency of lysogenization.