Structural elucidation of N-terminal post-translational modifications by mass spectrometry: application to chicken enolase and the alpha- and beta-subunits of bovine mitochondrial F1-ATPase

Peptides generated from enzymatic hydrolysis of chicken enolase and the alpha- and beta-subunits of bovine F1-ATPase were analyzed by mass spectrometry to determine the nature of their modified N-termini. In the case of chicken enolase, a peptide was isolated from a Staphylococcus aureus proteinase digest by HPLC and analyzed directly by fast atom bombardment mass spectrometry (FABMS). In conjunction with mass spectral evidence obtained from the methyl ester derivative and a secondary tryptic peptide, a structure is proposed containing an N-acetyl serine at the N-terminus. The alpha-subunit of bovine mitochondrial ATPase was chromatographed by HPLC after S. aureus proteinase digestion and a single peak was analyzed on the basis of predicted retention times. A Mr 716 was determined by FABMS and pyrrolidone carboxylic acid was deduced on the basis of its amino acid composition and partial Edman sequence data. The beta-subunit of ATPase produced a series of closely eluting peaks on HPLC after limited digestion with trypsin of the alpha 2 beta 2 complex. These peptides were analyzed by both Edman degradation and FABMS. These data showed the N-terminus to be frayed with N-terminal sequences beginning in pyro-Glu-Ala-Ser, Gln-Ala-Ser, Glu-Ala-Ser, Ala-Ser, and Ser but with no N-acetyl-Ser as was previously thought.     

Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.The etiologies of acute liver injury are diverse and its overall public health burden is considerable. Liver injury from acetaminophen (APAP) overdose is the most common cause of death from over-the-counter drugs and is the leading cause of acute liver failure in the developed world.^{1, 2, 3} Hepatic dysfunction from autoimmune hepatitis has a prevalence of 10–20/100 000.^{4, 5} Other etiologies of acute liver failure include idiosyncratic reaction to medications such as tetracycline, severe viral or alcoholic hepatitis, acute fatty liver of pregnancy, and idiopathic causes. Clinical complications resulting from liver failure include hepatic encephalopathy, impaired protein synthesis, and coagulopathies. Moreover, there are no effective means to reverse liver failure once advanced disease sets in – regardless of etiology – and transplantation frequently remains the only option for survival.⁶Concanavalin-A (ConA) is a lectin derived from the jack-bean plant with a unique ability to induce hepatitis in a well-described murine model of acute hepatic injury. ConA stimulates mouse CD4⁺ T-cell subsets to mediate insult to hepatocytes. The resulting cytokine release can further lead to recruitment and activation of innate inflammatory mediators, which perpetuate an insidious cycle of inflammation and hepatocellular injury.^{7, 8, 9}APAP is a widely used analgesic and antipyretic. Although usually considered safe at therapeutic doses, at higher doses APAP causes acute liver failure characterized by centrilobular hepatic necrosis.^{1, 10} At therapeutic doses, >90% of APAP is metabolized by glucuronidation and sulphation and its metabolites are excreted via the renal system. Of the remaining APAP, roughly 2% is excreted intact in the urine, and approximately 8% is metabolized by the cytochrome P450 system to N-acetyl-p-benzo-quinone imine (NAPQI), which is highly reactive.^{11, 12} Hepatic glutathione (GSH) then induces the formation of a safely excretable APAP-protein adduct. However, at toxic doses of APAP, GSH becomes depleted and NAPQI is able to exert harmful effects by forming covalent bonds with mitochondrial proteins, inhibiting the Ca²⁺-Mg²⁺-ATPase and inducing mitochondrial dysfunction.^{1, 2} This disturbance leads to a decrease in ATP synthesis, disruption of cellular membrane, and eventually hepatocyte death.¹³Although GSH depletion and the resulting toxic metabolites are prerequisites for APAP hepatotoxicity, there is evidence that the severity of liver injury may depend on subsequent participation of innate immunity.^{10, 14, 15, 16} In particular, APAP-induced injury has been reported to be contingent on activation of the NLRP3 inflammasome via DAMPs released from injured hepatocytes. Inflammasome activation cleaves Caspase 1 inducing IL-1β release and galvanizing intrahepatic neutrophils and inflammatory monocytes, which exacerbate injury.¹⁷ However, alternate studies using transgenic mice suggest that NLRP3 inflammasome is largely dispensable for APAP toxicity.¹⁸ Thus the role of inflammasome activation in APAP toxicity is controversial and may be dependent on the precise experimental conditions or strain of mice employed.Apoptosis and necrosis are classically understood processes of cell death that denote either organized Caspase 8-dependent programmed cell death or non-programmed disorganized death, respectively. In contrast to necrosis, which leads to the release of DAMPs and sustains inflammation, apoptosis produces cell fragments called apoptotic bodies, which phagocytic cells are able to engulf before the contents of the cell can spill out onto the surrounding space and activate innate immunity. ‘Necroptosis'' is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. Necroptosis requires the co-activation of RIP1 and RIP3 kinases. Both in vitro and in vivo investigations have suggested that APAP can induce cellular demise via necrosis or Caspase 8-dependent apoptosis, which is determined, in part, by ATP availability from glycolysis.¹⁹ Zhang et al.²⁰ recently confirmed that RIP1 is necessary in APAP-induced necroptosis. Similarly, Takemoto et al.²¹ showed that RIP1 inhibition protects against reactive oxygen species (ROS)-induced hepatotoxicity in APAP-induced acute liver injury. Further, a recent report suggested that selective inhibition of RIP3 using the anticancer drug Dabrafenib alleviates APAP injury.²²In the ConA model of acute liver injury, experiments using apoptosis-resistant mice expressing mutant FADD revealed that ConA alone induced primarily necrotic cell death, whereas ConA combined with d-galactosamine induced apoptosis and necrotic cell death.²³ Zhou et al.²⁴ reported that Necrostatin-1 (Nec-1) prevents autoimmune hepatitis in mice via RIP1- and autophagy-related pathways. Another recent report investigated the role of RIP1, RIP3, and PARP-1 in murine autoimmune hepatitis. This study found that in cases where death of mouse hepatocytes is dependent on TRAIL and NKT cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by RIP1 or PARP-1 inhibitors.²⁵ Our goal in the current study was to investigate, in parallel, the effects of RIP1 and RIP3 blockade in diverse models of acute liver injury. Our work suggests that modulating necroptosis may have divergent effects, depending on the etiology of hepatic injury and the specific component of the necrosome being targeted.     

A novel sex-determining QTL in Nile tilapia (Oreochromis niloticus)

Background

Fish species often exhibit significant sexual dimorphism for commercially important traits. Accordingly, the control of phenotypic sex, and in particular the production of monosex cultures, is of particular interest to the aquaculture industry. Sex determination in the widely farmed Nile tilapia (Oreochromis niloticus) is complex, involving genomic regions on at least three chromosomes (chromosomes 1, 3 and 23) and interacting in certain cases with elevated early rearing temperature as well. Thus, sex ratios may vary substantially from 50%.

Results

This study focused on mapping sex-determining quantitative trait loci (QTL) in families with skewed sex ratios. These included four families that showed an excess of males (male ratio varied between 64% and 93%) when reared at standard temperature (28°C) and a fifth family in which an excess of males (96%) was observed when fry were reared at 36°C for ten days from first feeding. All the samples used in the current study were genotyped for two single-nucleotide polymorphisms (rs397507167 and rs397507165) located in the expected major sex-determining region in linkage group 1 (LG 1). The only misassigned individuals were phenotypic males with the expected female genotype, suggesting that those offspring had undergone sex-reversal with respect to the major sex-determining locus. We mapped SNPs identified from double digest Restriction-site Associated DNA (ddRAD) sequencing in these five families. Three genetic maps were constructed consisting of 641, 175 and 1,155 SNPs from the three largest families. QTL analyses provided evidence for a novel genome-wide significant QTL in LG 20. Evidence was also found for another sex-determining QTL in the fifth family, in the proximal region of LG 1.

Conclusions

Overall, the results from this study suggest that these previously undetected QTLs are involved in sex determination in the Nile tilapia, causing sex reversal (masculinisation) with respect to the XX genotype at the major sex-determining locus in LG 1.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1383-x) contains supplementary material, which is available to authorized users.     

Multiple cytokines are released when blood from patients with tuberculosis is stimulated with Mycobacterium tuberculosis antigens

Background

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.

Methods

Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.

Results

Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.

Conclusions

Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Investigations of Homologous Recombination Pathways and Their Regulation

The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.     

Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli

Penicillin-binding protein 5 (PBP5) is a DD-carboxypeptidase, which cleaves the terminal D-alanine from the muramyl pentapeptide in the peptidoglycan layer of Escherichia coli and other bacteria. In doing so, it varies the substrates for transpeptidation and plays a key role in maintaining cell shape. In this study, we have analyzed the oligomeric state of PBP5 in detergent and in its native environment, the inner membrane. Both approaches indicate that PBP5 exists as a homo-oligomeric complex, most likely as a homo-dimer. As the crystal structure of the soluble domain of PBP5 (i.e., lacking the membrane anchor) shows a monomer, we used our experimental data to generate a model of the homo-dimer. This model extends our understanding of PBP5 function as it suggests how PBP5 can interact with the peptidoglycan layer. It suggests that the stem domains interact and the catalytic domains have freedom to move from the position observed in the crystal structure. This would allow the catalytic domain to have access to pentapeptides at different distances from the membrane.