Caspase proteolysis of the cohesin component RAD21 promotes apoptosis

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp(279) by caspases-3 and -7 in vitro to generate two major proteolytic products of approximately 65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.     

Cutting edge: susceptibility to psoriatic arthritis: influence of activating killer Ig-like receptor genes in the absence of specific HLA-C alleles

NK cell activity is partially controlled through interactions between killer Ig-like receptors (KIR) on NK cells and their respective HLA class I ligands. Independent segregation of HLA and KIR genes, along with KIR specificity for particular HLA allotypes, raises the possibility that any given individual may express KIR molecules for which no ligand is present. Inhibitory receptor genes KIR2DL2/3 and KIR2DL1 were present in nearly all subjects sampled in this study, whereas their respective activating homologs, KIR2DS2 and KIR2DS1, are each present in about half of the subjects. In this work we report that subjects with activating KIR2DS1 and/or KIR2DS2 genes are susceptible to developing psoriatic arthritis, but only when HLA ligands for their homologous inhibitory receptors, KIR2DL1 and KIR2DL2/3, are missing. Absence of ligands for inhibitory KIRs could potentially lower the threshold for NK (and/or T) cell activation mediated through activating receptors, thereby contributing to pathogenesis of psoriatic arthritis.     

The innate immune response differs in primary and secondary Salmonella infection

This study examines innate immunity to oral Salmonella during primary infection and after secondary challenge of immune mice. Splenic NK and NKT cells plummeted early after primary infection, while neutrophils and macrophages (Mphi) increased 10- and 3-fold, respectively. In contrast, immune animals had only a modest reduction in NK cells, no loss of NKT cells, and a slight increase in phagocytes following secondary challenge. During primary infection, the dominant sources of IFN-gamma were, unexpectedly, neutrophils and Mphi, the former having intracellular stores of IFN-gamma that were released during infection. IFN-gamma-producing phagocytes greatly outnumbered IFN-gamma-producing NK cells, NKT cells, and T cells during the primary response. TNF-alpha production was also dominated by neutrophils and Mphi, which vastly outnumbered NKT cells producing this cytokine. Neither T cells nor NK cells produced TNF-alpha early during primary infection. The TNF-alpha response was reduced in a secondary response, but remained dominated by neutrophils and Mphi. Moreover, no significant IFN-gamma production by Mphi was associated with the secondary response. Indeed, only NK1.1(+) cells and T cells produced IFN-gamma in these mice. These studies provide a coherent view of innate immunity to oral Salmonella infection, reveal novel sources of IFN-gamma, and demonstrate that immune status influences the nature of the innate response.     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.     

Expression,purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.     

Consistent patterns in the development and immunodominance of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses following acute HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses generated during acute infection play a critical role in the initial control of viremia. However, little is known about the viral T-cell epitopes targeted during acute infection or about their hierarchy in appearance and relative immunodominance over time. In this study, HIV-1-specific CD8+ T-cell responses in 18 acutely infected individuals expressing HLA-A3 and/or -B7 were characterized. Detailed analysis of CD8 responses in one such person who underwent treatment of acute infection followed by reexposure to HIV-1 through supervised treatment interruptions (STI) revealed recognition of only two cytotoxic T-lymphocyte (CTL) epitopes during symptomatic acute infection. HIV-1-specific CD8+ T-cell responses broadened significantly during subsequent exposure to the virus, ultimately targeting 27 distinct CTL epitopes, including 15 different CTL epitopes restricted by a single HLA class I allele (HLA-A3). The same few peptides were consistently targeted in an additional 17 persons expressing HLA-A3 and/or -B7 during acute infection. These studies demonstrate a consistent pattern in the development of epitope-specific responses restricted by a single HLA allele during acute HIV-1 infection, as well as persistence of the initial pattern of immunodominance during subsequent STI. In addition, they demonstrate that HIV-1-specific CD8+ T-cell responses can ultimately target a previously unexpected and unprecedented number of epitopes in a single infected individual, even though these are not detectable during the initial exposure to virus. These studies have important implications for vaccine design and evaluation.     

Favorable and unfavorable HLA class I alleles and haplotypes in Zambians predominantly infected with clade C human immunodeficiency virus type 1

The setpoint of viral RNA concentration (viral load [VL]) during chronic human immunodeficiency virus type 1 (HIV-1) infection reflects a virus-host equilibration closely related to CD8(+) cytotoxic T-lymphocyte (CTL) responses, which rely heavily on antigen presentation by the human major histocompatibility complex (MHC) (i.e., HLA) class I molecules. Differences in HIV-1 VL among 259 mostly clade C virus-infected individuals (137 females and 122 males) in the Zambia-UAB HIV Research Project (ZUHRP) were associated with several HLA class I alleles and haplotypes. In particular, general linear model analyses revealed lower log(10) VL among those with HLA allele B*57 (P = 0.002 [without correction]) previously implicated in favorable response and in those with HLA B*39 and A*30-Cw*03 (P = 0.002 to 0.016); the same analyses also demonstrated higher log(10) VL among individuals with A*02-Cw*16, A*23-B*14, and A*23-Cw*07 (P = 0.010 to 0.033). These HLA effects remained strong (P = 0.0002 to 0.075) after adjustment for age, gender, and duration of infection and persisted across three orders of VL categories (P = 0.001 to 0.084). In contrast, neither B*35 (n = 15) nor B*53 (n = 53) showed a clear disadvantage such as that reported elsewhere for these closely related alleles. Other HLA associations with unusually high (A*68, B*41, B*45, and Cw*16) or low (B*13, Cw*12, and Cw*18) VL were either unstable or reflected their tight linkage respecting disequilibria with other class I variants. The three consistently favorable HLA class I variants retained in multivariable models and in alternative analyses were present in 30.9% of subjects with the lowest (<10,000 copies per ml) and 3.1% of those with the highest (>100,000) VL. Clear differential distribution of HLA profiles according to level of viremia suggests important host genetic contribution to the pattern of immune control and escape during HIV-1 infection.     

Induction of mucosal protection against primary, heterologous simian immunodeficiency virus by a DNA vaccine

An effective vaccine against human immunodeficiency virus (HIV) should protect against mucosal transmission of genetically divergent isolates. As a safe alternative to live attenuated vaccines, the immunogenicity and protective efficacy of a DNA vaccine containing simian immunodeficiency virus (SIV) strain 17E-Fr (SIV/17E-Fr) gag-pol-env was analyzed in rhesus macaques. Significant levels of cytotoxic T lymphocytes (CTL), but low to undetectable serum antibody responses, were observed following multiple immunizations. SIV-specific mucosal antibodies and CTL were also detected in rectal washes and gut-associated lymphoid tissues, respectively. Vaccinated and naive control monkeys were challenged intrarectally with SIV strain DeltaB670 (SIV/DeltaB670), a primary isolate whose env is 15% dissimilar to that of the vaccine strain. Four of seven vaccinees were protected from infection as determined by the inability to identify viral RNA or DNA sequences in the peripheral blood and the absence of anamnestic antibody responses postchallenge. This is the first report of mucosal protection against a primary pathogenic, heterologous isolate of SIV by using a commercially viable vaccine approach. These results support further development of a DNA vaccine for protection against HIV.     

Elevated chemokine responses are maintained in lungs after clearance of viral infection

We observed two patterns of chemokine expression in the lungs of mice infected with murine gammaherpesvirus 68: peaks of chemokine expression correlated with or occurred after the peak of viral gene expression. Chemokine expression remained elevated through 29 days postinfection.     

Relationship of Mcl-1 isoforms,ratio p21WAF1/cyclin A,and Jun kinase phosphorylation to apoptosis in human breast carcinomas

Full length Mcl-1 is an anti-apoptotic protein consisting of two closely migrating 42/40kDa species. We now investigated the relationship of these isoforms to the expression of cell cycle stimulatory (cyclin A) and inhibitory (p21WAF1) proteins and to the induction of apoptosis in wt p53 MCF-7 and mutant p53 SKBR3 human breast carcinomas. The latter cells exhibited lower 42kDa Mcl-1, higher expression of cyclin A relative to that of p21WAF1, and apoptosis in response to okadaic acid, a phosphatase 1/2A inhibitor. The proteasome inhibitor MG-115 selectively increased expression of the 40kDa Mcl-1 isoform and induced p21WAF1, but also promoted preferential apoptosis in SKBR3 cells. Neither okadaic acid nor MG-115 caused comparable effects in MCF-7 cells. However, vanadate or acetyl furanonaphthoquinone induced the 40kDa Mcl-1 and greater Jun kinase (JNK) phosphorylation without apoptosis-associated PARP fragmentation in MCF-7 cells. Our data suggest that the higher susceptibility of SKBR3 cells to undergo apoptosis may be partly due to their greater proliferative potential (cyclin A), low expression of the anti-apoptotic 42kDa Mcl-1 isoform, and suboptimal JNK activation in response to stress.