Host Insect Inhibitor-of-Apoptosis SfIAP Functionally Replaces Baculovirus IAP but Is Differentially Regulated by Its N-Terminal Leader

The inhibitor-of-apoptosis (IAP) proteins encoded by baculoviruses bear a striking resemblance to the cellular IAP homologs of their invertebrate hosts. By virtue of the acquired selective advantage of blocking virus-induced apoptosis, baculoviruses may have captured cellular IAP genes that subsequently evolved for virus-specific objectives. To compare viral and host IAPs, we defined antiapoptotic properties of SfIAP, the principal cellular IAP of the lepidopteran host Spodoptera frugiperda. We report here that SfIAP prevented virus-induced apoptosis as well as viral Op-IAP3 (which is encoded by the Orgyia pseudotsugata nucleopolyhedrovirus) when overexpressed from the baculovirus genome. Like Op-IAP3, SfIAP blocked apoptosis at a step prior to caspase activation. Both of the baculovirus IAP repeats (BIRs) were required for SfIAP function. Moreover, deletion of the C-terminal RING motif generated a loss-of-function SfIAP that interacted and dominantly interfered with wild-type SfIAP. Like Op-IAP3, wild-type SfIAP formed intracellular homodimers, suggesting that oligomerization is a functional requirement for both cellular and viral IAPs. SfIAP possesses a ∼100-residue N-terminal leader domain, which is absent among all viral IAPs. Remarkably, deletion of the leader yielded a fully functional SfIAP with dramatically increased protein stability. Thus, the SfIAP leader contains an instability motif that may confer regulatory options for cellular IAPs that baculovirus IAPs have evolved to bypass for maximal stability and antiapoptotic potency. Our findings that SfIAP and viral IAPs have common motifs, share multiple biochemical properties including oligomerization, and act at the same step to block apoptosis support the hypothesis that baculoviral IAPs were derived by acquisition of host insect IAPs.Apoptosis is a prevalent host cell response to virus infection. Representing an important antivirus defense, apoptotic cell death can limit multiplication and virus dissemination in the host. Thus, the mechanisms by which a host organism detects a viral intruder and initiates the apoptotic response are critical to the outcome of the infection for both the host and virus. The cellular inhibitor-of-apoptosis (IAP) proteins are important candidates for sensing virus infection and determining cell fate by virtue of their central position in the apoptosis pathway (reviewed in references 35, 36, and 44). Affirming their importance in regulation of apoptosis, IAPs are encoded by multiple DNA viruses, including baculoviruses, entomopoxviruses, iridoviruses, and African swine fever virus (reviewed in 3). Nonetheless, the molecular mechanisms by which viral IAPs regulate virus-induced apoptosis and how they biochemically differ from cellular IAPs are poorly understood.The IAPs were first discovered in baculoviruses because of their capacity to prevent virus-induced apoptosis and thereby facilitate virus multiplication (4, 8). The baculovirus IAPs bear a striking resemblance to the cellular IAPs carried by the host insects that they infect. Cellular IAPs are a highly conserved family of survival factors that regulate developmental and stress-induced apoptosis, as well as inflammation, the cell cycle, and other signaling processes (35, 38, 44). Importantly, misregulation or overexpression of IAPs is associated with neoplasia and tumor chemoresistance (24, 49). The IAPs are defined by the presence of one or more ∼80-residue baculovirus IAP repeat (BIR) domains. The BIRs consist of a conserved Zn²⁺-coordinating arrangement of Cys and His residues (CCHC) that interact with diverse proteins, including the cysteinyl aspartate-specific proteases called caspases that execute apoptosis (reviewed in 16 and 37). The antiapoptotic activity of some, but not all, IAPs is derived from their ability to bind and neutralize caspases (reviewed in 35 and 44). The BIRs also interact with proapoptotic factors that contain IAP binding motifs (IBMs). IBM-containing factors have the capacity to bind and dissociate the IAP-caspase complex, thereby liberating active caspases to execute apoptosis (16, 35, 36, 48). Many IAPs, including viral IAPs, also possess a C-terminal RING domain, which is a Zn²⁺-coordinating motif with E3-ubiquitin ligase activity, which can contribute to antiapoptotic activity (48).The best-studied baculovirus IAP is Op-IAP3, which is encoded by Orgyia pseudotsugata nucleopolyhedrovirus. This small IAP (268 residues) contains two BIRs and a C-terminal RING (Fig. ​(Fig.1A).1A). Both BIRs are required for Op-IAP3 antiapoptotic activity (19, 50, 53). Truncation of the Op-IAP3 RING creates a loss-of-function dominant inhibitor (19). Op-IAP3''s capacity to form a complex with this RING-lacking (RINGless) dominant inhibitor and with itself suggests that oligomerization is necessary for IAP function. Upon overexpression, Op-IAP3 blocks apoptosis triggered by diverse signals in cells from certain insects and mammals, suggesting that it acts through a conserved mechanism (7, 11, 15, 33, 51, 54, 56). In the baculovirus host moth Spodoptera frugiperda (Lepidoptera: Noctuidae), Op-IAP3 prevents apoptosis by blocking the activation of effector caspases (25, 32, 40). However, in contrast to host insect IAPs, Op-IAP3 fails to inhibit active caspases (45, 51, 54). Thus, the host cell target(s) and the mechanism by which they are neutralized by this viral IAP remain unclear.Open in a separate windowFIG. 1.SfIAP structure and mutagenesis. (A) Viral and cellular IAPs. Viral Op-IAP3 (268 residues) and SfIAP (377 residues) each contain two BIR motifs (black boxes) and an E3 ligase RING domain (cross-hatched box). Each representing a potential start site, four methionines (M1 to M4) exist in the N-terminal leader of SfIAP. (B) SfIAP^M4 mutations. SfIAP^M4 (281 residues) begins with the M4 methionine. SfIAP^M4ΔR (227 residues) lacks the C-terminal RING. Amino acid substitutions of Zn-coordinating residues are indicated. An epitope tag (HA) was inserted at the N terminus. (C) Marker rescue assay. The antiapoptotic activity of wild-type or mutated forms of SfIAP^M4 was assayed by virus marker rescue in which replication of p35-deficient vΔp35/lacZ was restored in proportion to the antiapoptotic activity of the mutated Sfiap gene acquired by integration of the SfIAP-encoding plasmid (2). Virus yields were determined by plaque assay using apoptosis-sensitive SF21 cells. Antiapoptotic activity is reported as the ratio of nonapoptotic, lacZ-expressing plaques produced by transfection of the indicated Sfiap to those produced by wild-type Sfiap. Values shown are the averages ± standard deviations obtained from triplicate transfections.Among the cellular IAPs, SfIAP from Spodoptera frugiperda is most closely related to viral Op-IAP3. SfIAP (Fig. ​(Fig.1A)1A) is 42% identical to Op-IAP3, with a higher degree of amino acid identity localized to its two BIRs and C-terminal RING (20). As the principal IAP in Spodoptera, SfIAP suppresses a constitutive push toward apoptosis (34); ablation of SfIAP leads to immediate apoptosis of cultured Spodoptera cells. Upon overexpression, SfIAP also rescues the multiplication of apoptosis-inducing baculoviruses and can prevent apoptosis in certain mammalian cell lines (20, 26). In contrast to viral Op-IAP3, SfIAP can bind and inhibit caspases, including Spodoptera frugiperda caspase-1 (Sf-caspase-1) and human caspase-9 (20, 45). Thus, despite their structural similarities, there exist fundamental differences in the biochemical activities of these two IAPs. Importantly, SfIAP fails to prevent baculovirus-induced apoptosis when produced at endogenous levels in permissive Spodoptera cells. Thus, it is expected that SfIAP also possesses regulatory motifs that respond to cellular signals triggered upon virus infection.SfIAP provides an unprecedented opportunity to investigate the functional and evolutionary relationships between host and viral IAPs and to test the intriguing hypothesis that viral IAPs were acquired by host gene capture (21). We have investigated the biochemical properties of SfIAP as a means to define its molecular mechanisms and to test its relatedness to viral IAPs. We report here that SfIAP shares many biochemical and functional features with viral IAPs. Like Op-IAP3, overexpressed SfIAP prevented virus-induced apoptosis at a step upstream of caspase activation by a mechanism that required BIR1, BIR2, and the RING. SfIAP formed a complex with itself and with a RINGless dominant inhibitor, suggesting that oligomerization is also required for function of cellular IAPs. Unlike viral IAPs, SfIAP possesses an N-terminal leader, which modulates intracellular SfIAP levels and may respond to apoptotic signals to regulate cell survival. Our data are consistent with a model in which baculoviruses acquired a host cell IAP and modified it for virus-specific needs, thereby increasing virus fitness by preventing virus-induced apoptosis.     

Novel function of cardiac protein kinase D1 as a dynamic regulator of Ca2+ sensitivity of contraction

Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated reticular pattern, suggesting the importance of PKD in Ca(2+) signaling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.     

Characterization of N-Linked Protein Glycosylation in Helicobacter pullorum

The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the −2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.Glycosylation is one of the most common protein modifications, and eukaryotes glycosylate many of their secreted proteins with asparagine or N-linked glycans. This process is thought to have diverse roles in protein folding, quality control, protein secretion, and sorting (13). Eukaryotic glycosylation takes place at the luminal side of the endoplasmic reticulum (ER) membrane, where a preassembled oligosaccharide is transferred from a lipid carrier to asparagine residues within an N-X-S/T consensus sequence, where X can be any amino acid except proline (19). The coupling of glycan to the protein takes place cotranslationally as nascent polypeptide chains cross the ER membrane via a translocon apparatus (5). This reaction involves a protein complex of at least eight subunits (49), with the STT3 protein (50, 52) apparently acting as the central enzyme in the process of N-linked protein glycosylation (29, 48). The STT3 protein consists of an amino terminus with multiple membrane-spanning domains and a carboxy-terminal region containing the highly conserved WWDYG amino acid sequence motif (15).The first prokaryotic glycoproteins were described for archaeal species over 30 years ago (26), and for some time it was thought that protein glycosylation was a eukaryotic and archaeal, but not a bacterial, trait. However, there are now many examples of protein glycosylation in species from the domain Bacteria. For example, general O-linked protein glycosylation systems in which functionally diverse sets of proteins are glycosylated via a single pathway have recently been identified in Neisseria and Bacteroides spp. (8, 21, 44). The most-well-characterized bacterial species with respect to protein glycosylation is the enteropathogen Campylobacter jejuni, which encodes an O-linked system that glycosylates the flagellin protein of the flagellar filament along with the first described bacterial N-linked glycosylation system (39).The C. jejuni N-linked glycosylation pathway is encoded by genes from a single protein glycosylation, or pgl, locus (38). The glycosylation reaction is thought to occur at the periplasmic face of the bacterial inner membrane mediated by the product of the STT3 orthologue pglB (46). The C. jejuni heptasaccharide glycan is assembled on a lipid carrier in the cytoplasm through the action of glycosyltransferases encoded by the pglA, pglC, pglH, pglJ, and pglI genes (11, 12, 24, 31). This lipid-linked oligosaccharide (LLO) is then “flipped” into the periplasm by the pglK gene product, or “flippase” (1), and transferred by PglB onto an asparagine residue within an extended D/E-X-N-X-S/T sequon (19). Many C. jejuni periplasmic and surface proteins of diverse function are N glycosylated (51), yet the function of glycosylation remains elusive. Unlike in eukaryotes, this process occurs posttranslationally, and the surface location of the sequon in folded proteins appears to be required for glycosylation (20).The C. jejuni pgl gene locus can be transferred into Escherichia coli, and the corresponding gene products will function to transfer the heptasaccharide onto asparagine residues of coexpressed C. jejuni glycoproteins as well as non-C. jejuni proteins containing the appropriately located acceptor sequon (19, 46). When alternative lipid-linked glycans are present, such as those involved in lipopolysaccharide biosynthesis, glycans with diverse structure can also be transferred onto proteins (7). Although there are limitations, particularly with regard to the apparent structural requirement for an acetamido group on the C-2 carbon of the reducing end sugar (7, 47), this is still a significant advance toward tractable in vivo systems for glycoconjugate synthesis. The identification and characterization of further bacterial PglB proteins with potentially diverse properties would considerably expand the utility of such systems. Data from genome sequencing indicate that pglB orthologues are found in species closely related to C. jejuni, such as Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis (40), as well as in the more distantly related species Wolinella succinogenes (2). These species are members of the phylogenetic grouping known as the epsilon subdivision of the Proteobacteria, or Epsilonproteobacteria, consisting of the well-established genera Campylobacter, Helicobacter, Arcobacter, and Wolinella, which are often associated with human and animal hosts, as well as a number of newly recognized groupings of environmental bacteria often found in sulfidic environments (3). However, not all species of Epsilonproteobacteria contain pglB orthologues, and until recently, all characterized Helicobacter species lacked pglB genes.Given the considerable interest in exploiting bacterial protein glycosylation, especially the C. jejuni N-linked glycosylation system, for generating glycoconjugates of biotechnological and therapeutic potential, the functional characterization of newly discovered pglB orthologues is a priority. In this report we describe the application of an in vitro oligosaccharyltransferase assay to investigate N-linked glycosylation initially in C. jejuni, where the utility of this approach was demonstrated, and then in Helicobacter pullorum, demonstrating that one of the two H. pullorum PglB enzymes is responsible for N-linked protein glycosylation with a pentasaccharide glycan.     

Understanding signal integration through targeted mutations of an adapter protein

Immunoreceptor engagement leads to the activation of multiple second messenger cascades, and integration of these pathways requires proper function of a number of adapter proteins. Although adapters possess no intrinsic enzymatic function, they nucleate the formation of multi-molecular protein complexes to support downstream signaling. Since adapters contain functionally distinct domains, intense investigation has been devoted to understanding how these regions act to integrate signals. This review describes the evolution of studies investigating one of these adapters, the SH2 domain-containing leukocyte protein of 76 kDa. Through utilizing biochemical, genetic and imaging techniques, a model has emerged describing how this adapter regulates signals resulting in complex immune responses.     

Developmental origins of diabetes: the role of oxidative stress

The "thrifty phenotype" hypothesis proposes that the fetus adapts to an adverse intrauterine milieu by optimizing the use of a reduced nutrient supply to ensure survival, but, by favoring the development of certain organs over that of others, this leads to persistent alterations in the growth and function of developing tissues. This concept has been somewhat controversial; however, recent epidemiological, clinical, and animal studies provide support for the developmental origins of disease hypothesis. Underlying mechanisms include reprogramming of the hypothalamic-pituitary-adrenal axis, islet development, and insulin signaling pathways. Emerging data suggest that oxidative stress and mitochondrial dysfunction may also play critical roles in the pathogenesis of type 2 diabetes in individuals who were growth retarded at birth.     

Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.     

The Src homology 2 domain-containing leukocyte protein of 76-kDa adaptor links integrin ligation with p44/42 MAPK phosphorylation and podosome distribution in murine dendritic cells

The Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. In this study, we report that SLP-76 is expressed in CD11c+ B220- dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76-deficient BMDCs adhere poorly to fibronectin, suggesting impaired integrin function. Consistent with impaired adhesion, cutaneous SLP-76-deficient DCs leave ear tissue at an elevated frequency compared with wild-type DCs. In addition, the pattern and distribution of actin-based podosome formation are visibly altered in BMDCs lacking SLP-76 following integrin engagement. SLP-76-deficient BMDCs manifest multiple signaling defects following integrin ligation, including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAPK (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hemopoietic cells may use unique molecular intermediates and mechanisms for regulating integrin signaling.