Molecular cloning and characterization of a receptor for activated protein kinase C1 (RACK1) from Chinese white shrimp; Fenneropenaeus chinensis

Receptor for activated C kinase 1 (RACK1), which has seven tandem WD40 domains, is a scaffolding protein. RACK1 plays different roles by binding to different partner proteins. It is involved in hormone signaling and development, and now some evidence indicates it may have a role in innate immunity. In this paper, RACK1 cDNA from Chinese white shrimp (FcRACK1) was identified. The full length of the FcRACK1 gene is 1037 bp, including a 30 bp 5′UTR, a 957 bp ORF encoding a 318 amino acid protein, and a 50 bp 3′UTR with the polyadenylation sequence AATAAA and a poly (A) tail. The FcRACK1 protein is characterized by seven WD40 repeat domains; the ending two amino acids of each WD40 domain are WK, WD, WN, WS, WD, WD, and WQ, respectively. The length of each domain is between 30 and 44 amino acids. Multiple alignments of RACK1s showed that RACK1s are highly conserved. RT-PCR showed that FcRACK1 could be detected in hemocytes, the heart, hepatopancreas, gills, stomach, intestine, and ovary. FcRACK1 in hemocytes was down-regulated after a 2 h WSSV challenge, and FcRACK1 in gills was up-regulated after a 2 h Vibrio challenge. FcRACK1 in ovary went down after a 12 h Vibrio challenge and then up-regulated at 24 h. FcRACK1 in ovary was first down-regulated at 2 h after a WSSV challenge and then up-regulated to the highest level at 6 h. It finally went down from 12 to 24 h. In hepatopancreas, FcRACK1 was also up-regulated by microbe challenge. Our results indicated its probable role in shrimp innate immunity.     

Liver injury in Wilson's disease: An immunohistochemical study

PurposeWilson's disease (WD) is an inherited disorder involving copper accumulation in the liver and brain. An important mechanism responsible for hepatocyte injury in WD is mitochondria destruction, although damage may also be caused by oxidative stress and lipid peroxidation.Patients/methodsThe study included 54 treated patients with WD without liver cirrhosis and 10 healthy controls. All patients had liver biopsy and immunohistochemical analysis of liver samples was performed using targeted staining for markers of mitochondrial injury (thioredoxin-2 [TRX2], cytochrome c oxidases subunit 2 [COX2], and cytochrome c oxidases complex IV subunit 4 isoform 1 [COX4-1]), of oxidative stress (peroxiredoxin-1 [PRDX1] and 8-hydroxyguanosine [8-OHdG]), and of lipid peroxidation (4-hydroxynonenal [4-HNE]).ResultsExpression, measured as mean strengths of intensity (SI) of immunohistochemical reactions per 5 fields of view, was significantly lower in patients with WD compared to controls for COX2 (2.9 vs 8.3), 8-OHdG (0.05 vs 3.8), TRX2 (4.9 vs 10.1), and PRDX1 (4.6 vs 10.1) (all P ?< ?10^?5). COX4-1 expression was undetected in patients with WD but detected in control specimens (8.1) (P ?< ?10^?5). 4-HNE was overexpressed in patients with WD compared to controls (10.1 vs 9.1; P ?< ?0.07).ConclusionsNegligible COX4-1 and low COX2 expression in liver specimens may serve as markers of inner mitochondrial membrane injury in treated patients with WD and early stages of liver fibrosis.     

Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation

The development of age-related proliferative disorders of the prostate gland is supported by transdifferentiation and cellular senescence processes in the stroma. Both processes are involved in remodeling of stromal tissue, as observed in benign prostatic hyperplasia (BPH), and in “reactive stroma” adjacent to prostate cancer (PCa). It has been assumed that TGF-β1 plays a key role in the aging prostate by inducing premature senescence and favoring myofibroblast differentiation. Therefore, we evaluated the stromal cell phenotypes of human primary adult prostatic fibroblasts (n = 3) and the molecular and cellular mechanisms of growth arrest after treatment with TGF-β1 and of in vitro cellular senescence. Microarray analysis, quantitative PCR, immunofluorescence and western blot revealed that cellular senescence and transdifferentiation of fibroblasts have distinct underlying mechanisms, pathways and gene and protein expression profiles in human PrSCs. In clear contrast to senescent cells, TGF-β1-treated cells morphologically transdifferentiated into myofibroblasts with dense cytoskeletal fibers and increased expression of smooth muscle cell α-actin, calponin and tenascin. TGF-β1 induced neither expression of senescence-associated markers nor genes involved in terminal growth arrest, such as senescence-associated beta-galactosidase and cyclin-dependent kinase (cdk) inhibitors p16^Ink4A and p21^Cip1 but increased p15^Ink4B protein expression. Differentiation inhibitor (Id-1) protein level down-regulation was observed under both conditions. Genes specifically up-regulated by transdifferentiation but not by cellular senescence of PrSCs were metalloproteinase 1 tissue inhibitor (Timp1), transgelin (Tagln), gamma 2 actin (Actg2), plasminogen activator inhibitor 1 (Serpinel), insulin-like growth factor binding protein 3 (Igfbp3), parathyroid hormone-like hormone (Pthlp), Tgfb-1, four and a half LIM domains 2 (Fhl-2), hydrogen peroxide-inducible clone 5 (Hic5) and cartilage oligomeric matrix protein (Comp). Other genes, such as Cdc28 protein kinase 1 (Cks1b), v-myb myeloblastosis viral oncogene homolog (MybL2), pyruvate kinase, muscle 2 (Pkm2) and Forkhead box M1 (FoxM1), were down-regulated only upon TGF-β1 treatment but not by cellular senescence. Pyruvate dehydrogenase kinase 3 (Pdk3) and connective tissue growth factor (Ctgf) were up-regulated and hyaluronan synthase 3 (Has3) down-regulated under both conditions. Moreover, GageC1, a prostate/testis-specific protein overexpressed in symptomatic BPH and PCa was induced in transdifferentiated stromal cells. Genes such as GageC1 could be promising targets for therapeutic inhibitors of stromal tissue remodeling and progression of BPH and PCa.     

Tumour necrosis factor receptors and apoptosis of alveolar macrophages during early infection with attenuated and virulent Mycobacterium bovis

Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1^−/− mice at day 3 after bacillus Calmette–Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner.     

The P-type ATPase Spf1 is required for endoplasmic reticulum functions and cell wall integrity in Candida albicans

Endoplasmic reticulum (ER) is crucial for protein folding, glycosylation and secretion in eukaryotic organisms. These important functions are supported by high levels of Ca²⁺ in the ER. We have recently identified a putative ER Ca²⁺ pump in Candida albicans, called Spf1, which plays key roles in maintenance of cellular Ca²⁺ homeostasis, morphogenesis and virulence. In this study, we purified Spf1 and confirmed that it is a P-type ATPase, suggesting its role in maintaining high levels of ER Ca²⁺. Disruption of SPF1 caused severe defects in glycosylation of the ER-localized protein Cdc101 and secretory acid phosphatase, and a decrease in expression of SEC61 which encodes an important ER protein. Moreover, the spf1Δ/Δ mutant showed increased sensitivity to cell wall stresses, abnormal cell wall composition, delayed cell wall reconstruction and decreased flocculation and adherence, indicating its defect in cell wall integrity (CWI). We also revealed that disruption of SPF1 has an impact on gene expression related to CWI and morphogenesis. This study provides evidence that Spf1, as a P-type ATPase, is essential for ER functions and consequent CWI, implicating a role of ER Ca²⁺ homeostasis in C. albicans physiology.     

Decrease in Tumor Necrosis Factor-α Receptor-Associated Death Domain Results from Ubiquitin-Dependent Degradation in Obstructive Renal Injury in Rats

Increased expression levels of tumor necrosis factor-α (TNFα) is involved in tubulointerstitial cell proliferation and apoptosis in obstructive renal injury. Two TNFα receptors (TNFRs), TNFR1 and TNFR2, are known to exist. On TNFα binding, TNFR1 recruits TNFR-associated death domain (TRADD), an assembly platform to mediate TNFR1 signaling. We investigated postreceptor TRADD regulation in rat kidneys with unilateral ureteral obstruction (UUO). Whereas UUO was associated with increased expression levels of TNFα, TNFR1, TNFR2, and TRADD mRNAs, it resulted in the marked decrease of TRADD protein levels (which appeared at day 1 and persisted thereafter) and a slight decrease in TNFR1 protein levels at days 7 and 14. Both ubiquitination and degradation of TRADD were increased in UUO kidneys, degradation of TRADD was stimulated by TNFα in HK-2 cells, and TRADD degradation was suppressed by proteasome inhibitor. Inhibition of TNFα by soluble TNFR2, etanercept, reduced significantly, although transiently, tubular and interstitial cell proliferation, fibronectin expression, and apoptosis in UUO kidneys, and also suppressed TRADD degradation. These data suggest that the decrease in TRADD resulting from enhanced ubiquitin-dependent degradation is involved in obstructive renal injury. Since TRADD is not incorporated into TNFR2-mediated TNFα signaling, the persistent decrease in TRADD, associated with a mild decrease in TNFR1 levels, may function, at least in part, to divert TNFα signals toward a TNFR2-mediated pathway in UUO kidneys.Unilateral ureteral obstruction (UUO) is a well-established model of experimental renal injury characterized by significant renal tubular dilatation, proliferation, apoptotic cell death, and followed by tubulointerstitial fibrosis.^1,2 In the kidney, cell proliferation is believed to be a central response to injury and culminates in the development of fibrotic renal damage.³ An imbalance between cell proliferation and apoptosis leads to unchecked apoptosis, resulting in progressive cell loss, renal tubular atrophy, and interstitial fibrosis.⁴ Tumor necrosis factor-α (TNFα) is a highly pleiotropic cytokine that induces diverse cellular responses ranging from proliferation and differentiation to activation of apoptosis.⁵ Overexpression of TNFα is reported to be involved in proliferation and apoptosis of renal tubular and interstitial cells in obstructive renal injury.^6,7,8 However, little is known about the postreceptor regulation of TNFα signaling in renal lesions.TNFα binds to TNF receptors (TNFR) to elicit its biological functions. There are two different cell-surface TNFRs; TNFR1 and TNFR2, which originate from separate gene products.⁹ On binding of TNFα, TNFR1 recruits the adaptor protein, TNFR associated death domain (TRADD), directly to its cytoplasmic death domain. In turn, TRADD serves as an assembly platform to diverge TNFR1 signaling. Interaction of TRADD with receptor interacting protein and TNF receptor associated factor 2 (TRAF2) leads to the activation of nuclear factor κB (NFκB).¹⁰ Furthermore, TRADD is also involved in the recruitment of Fas-associated protein with death domain, resulting in the initiation of apoptosis through activation of the caspase-8/3 cascade.¹¹On the other hand, the precise mechanism of TNFR2-mediated signaling is not fully elucidated. One report demonstrated that the binding of TNFα to TNFR2 recruits TRAF2 and induces NFκB activation.¹² However, it was also shown that the binding of TNFα to TNFR2 causes ubiquitin-dependent degradation of TRAF2, resulting in the suppression of NFκB activation through the inhibition of TRADD, receptor interacting protein, and TRAF2 complex formation, and finally leading to TNFR1-mediated TNF-α signaling toward the pro-apoptotic direction.¹³At present, the differential contribution of TNFR1- and TNFR2-mediated TNFα signaling is not fully elucidated in renal lesions. Ramesh et al⁹ reported that renal injury induced by cisplatin was less severe in TNFR2-deficient mice than TNFR1-deficient mice. In contrast, Guo et al¹ reported that the renal lesions in UUO mice were less severe in TNFR1 knockout mice compared with TNFR2 knockout mice. There is no report on the involvement and regulation of TRADD, an assembly platform to diverge TNFR1 signaling, in the development of renal lesions. In the present study, we investigated the postreceptor regulation of TRADD in the UUO rat kidneys. The effect of TNFα inhibition by etanercept, a soluble TNFR2, was also studied in UUO rat kidneys.     

Pharmacokinetics and biochemical properties of human pro-urokinase variants carrying the deletion in epidermal growth factor-like domain

Two variants of pro-urokinase (pro-UK), Δ_11–32pro-UK lacking the first and the second loops (Cys¹¹Asn³²) within the EGF-like domain and δ_33–42pro-UK lacking the third loop (Cys³³Cys⁴²), were prod in Chinese hamster ovary cells. They were compared with natural pro-UK (n-pro-UK) and Δ_10–42pro-UK lacking entire EGF-like domain (Hiramatsu et al¹) in terms of clearance rate and biochemical properties.Fibrinolytic activity half-lives of these mutants in rat circulation after bolus administration were 7.1 min for Δ_11–32pro-UK and 6.8 min for Δ_33–42pro-UK, which were about 3.5 times longer than that of n-pro-UK (2.0 min) and slightly shorter than that of Δ_10–42pro-UK (7.6 min). The amidolytic activity of these deletion mutants were almost the same as n-pro-UK in terms of both Km and k_cat (3.4–4.O × 10⁻⁴ M, and 8.4–10.O × 10³ min⁻¹ respectively), suggesting that the protease domain was not affected by the deletion in EGF-like domain. It was also found that the binding ability to fibrin-celite was decreased by the absence of the third loop of the EGF-like domain. In addition, conversion from the single chain form to the two chain form by the action of plasmin was slowed in proportion to the size of the deletion in the EGF-like domain.     

TNFR1 plays a critical role in the control of severe HSV-1 encephalitis

Herpes simplex virus-1 (HSV-1) is a pathogen for humans that may cause severe encephalitis. Tumor necrosis factor α (TNF-α) plays a role in several viral diseases of the central nervous system (CNS). The classic proinflammatory activities of TNF-α are mediated mainly through activation of the receptor 1 for TNF-α (TNFR1). However, when HSV-1 is inoculated in the periphery, TNF-α seems to protect C57Bl/6 mice against encephalitis by a mechanism independent of TNFR1. This study aims to investigate the role of TNFR1 in HSV-1 encephalitis induced by the inoculation of the virus into the brain. Wild-type C57BL/6 (WT) and TNFR1^−/− were inoculated with 10² plaque-forming units of HSV-1 by the intracranial route. Infection with HSV-1 was lethal in TNFR1^−/− mice in early times after infection. TNFR1^−/− mice had reduced expression of the chemokines CCL3 and CCL5, and decreased leukocyte adhesion in the brain vasculature compared to WT mice 4 days post-infection (dpi). At this time point TNFR1^−/− infected mice also had higher HSV-1 viral replication and more injuries in the brain, especially in the hippocampus. In conclusion, TNFR1 seems to play a relevant role in the control of viral replication in the CNS when HSV-1 is inoculated by intracranial route.     

A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants

PurposeDNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care.MethodsWe report an E. coli–based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins.ResultsFirst, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses.ConclusionThis simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.     

Inactivation of DRG1, encoding a translation factor GTPase,causes a recessive neurodevelopmental disorder

PurposeDevelopmentally regulated Guanosine-5'-triphosphate-binding protein 1 (DRG1) is a highly conserved member of a class of GTPases implicated in translation. Although the expression of mammalian DRG1 is elevated in the central nervous system during development, and its function has been implicated in fundamental cellular processes, no pathogenic germline variants have yet been identified. Here, we characterize the clinical and biochemical consequences of DRG1 variants.MethodsWe collate clinical information of 4 individuals with germline DRG1 variants and use in silico, in vitro, and cell-based studies to study the pathogenicity of these alleles.ResultsWe identified private germline DRG1 variants, including 3 stop-gained p.Gly54¹, p.Arg140¹, p.Lys263¹, and a p.Asn248Phe missense variant. These alleles are recessively inherited in 4 affected individuals from 3 distinct families and cause a neurodevelopmental disorder with global developmental delay, primary microcephaly, short stature, and craniofacial anomalies. We show that these loss-of-function variants (1) severely disrupt DRG1 messenger RNA/protein stability in patient-derived fibroblasts, (2) impair its GTPase activity, and (3) compromise its binding to partner protein ZC3H15. Consistent with the importance of DRG1 in humans, targeted inactivation of mouse Drg1 resulted in preweaning lethality.ConclusionOur work defines a new Mendelian disorder of DRG1 deficiency. This study highlights DRG1’s importance for normal mammalian development and underscores the significance of translation factor GTPases in human physiology and homeostasis.     

Shedding of Tumor Necrosis Factor Receptor 1 Induced by Protein A Decreases Tumor Necrosis Factor Alpha Availability and Inflammation during Systemic Staphylococcus aureus Infection

Staphylococcus aureus infections are an important public health concern due to their increasing incidence and high rates of mortality. The success of S. aureus as a pathogen is highly related to its enormous capacity to evade the host immune response. The critical role of tumor necrosis factor alpha (TNF-α) in the initial host defense against systemic staphylococcal infection has been demonstrated in experimental models and may partially explain the lack of significant benefits observed in clinical trials attempting to neutralize this cytokine in septic patients. S. aureus protein A plays a key role in regulating inflammation through its ability to bind and signal through the TNF-α receptor 1 (TNFR1). In this study, we demonstrate that S. aureus, via protein A-mediated signaling, induces early shedding of TNFR1, which precedes the secretion of TNF-α in vitro and in vivo. The results obtained using a protein A-deficient mutant and tnfr1^−/− mice strongly suggest that the increased levels of soluble TNFR1 present during experimental S. aureus infection may neutralize circulating TNF-α and impair the host inflammatory response. Early shedding of TNFR1 induced by protein A may constitute a novel mechanism by which S. aureus subverts the host immune response.     

Molecular cloning and expression of TLR in the Eisenia andrei earthworm

Toll-like receptors (TLRs) play an important role in defense responses to pathogens in invertebrates. Here we characterize the first TLR isolated from an oligochaete annelid, namely, Eisenia andrei (EaTLR) and show its expression pattern. The full-length EaTLR cDNA consists of 2615 bp encoding a putative protein of 675 amino acids. The predicted amino acid sequence comprises of an extracellular domain containing 31 amino acid signal peptide and seven leucine-rich repeats (LRR), capped with cysteine-rich N- and C-terminal LRRs followed by a transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). TIR domains of twenty individual earthworms were sequenced and the variability suggesting the presence of a high number of TLR genes in the genome of E. andrei was observed. Phylogenetic analysis revealed the highest similarity of EaTLR with polychaete annelid, Capitella teleta and TLRs of mollusks and echinoderms. Finally, the highest constitutive expression of EaTLR was observed in the digestive tract. Gene expression was significantly increased in coelomocytes of E. andrei after the challenge with Gram-positive bacteria.     

Man1, an inner nuclear membrane protein,regulates left–right axis formation by controlling nodal signaling in a node‐independent manner

Man1, an inner nuclear membrane protein, regulates transforming growth factor β signaling by interacting with receptor‐associated Smads. In Man1‐deficient (Man1^Δ/Δ) embryos, vascular remodeling is perturbed by misregulation of Smad activity. Here, we show that Man1^Δ/Δ embryos exhibit abnormal heart morphogenesis including the looping abnormality. We searched for the molecular basis underlying the heart abnormalities and found that the left side‐specific genes responsible for left–right (LR) asymmetry, Nodal, Lefty2, and Pitx2, were expressed bilaterally in the lateral plate mesoderm and that their expression was enhanced significantly in mutants. Notably, Lefty1, a marker for the midline barrier, was maintained in Man1^Δ/Δ mutants. Crossing Man1^Δ/+ with Nodal hypomorphs (Nodal^neo/+), in which Nodal signaling in the node is disrupted, to generate double homozygous embryos (Man1^Δ/Δ; Nodal^neo/neo) revealed that the bilateral Nodal was retained in Man1^Δ/Δ; Nodal^neo/neo embryos. These results suggest that Man1 regulates LR asymmetry by controlling Nodal signaling in a node‐independent manner. Developmental Dynamics 237:3565–3576, 2008. © 2008 Wiley‐Liss, Inc.     

Complete sequence of RNA1 of grapevine Anatolian ringspot virus

The nucleotide sequence of RNA1 of grapevine Anatolian ringspot virus (GARSV), a nepovirus of subgroup B, was determined from cDNA clones. It is 7,288 nucleotides in length excluding the 3′ terminal poly(A) tail and contains a large open reading frame (ORF), extending from nucleotides 272 to 7001, encoding a polypeptide of 2,243 amino acids with a predicted molecular mass of 250 kDa. The primary structure of the polyprotein, compared with that of other viral polyproteins, revealed the presence of all the characteristic domains of members of the order Picornavirales, i.e., the NTP-binding protein (1B^Hel), the viral genome-linked protein (1C^VPg), the proteinase (1D^Prot), the RNA-dependent RNA polymerase (1E^Pol), and of the protease cofactor (1A^Pro-cof) shared by members of the subfamily Comovirinae within the family Secoviridae. The cleavage sites predicted within the polyprotein were found to be in agreement with those previously reported for nepoviruses of subgroup B, processing from 1A to 1E proteins of 67, 64, 3, 23 and 92 kDa, respectively. The RNA1-encoded polyprotein (p1) shared the highest amino acid sequence identity (66 %) with tomato black ring virus (TBRV) and beet ringspot virus (BRSV). The 5′- and 3′-noncoding regions (NCRs) of GARSV-RNA1 shared 89 % and 95 % nucleotide sequence identity respectively with the corresponding regions in RNA2. Phylogenetic analysis confirmed the close relationship of GARSV to members of subgroup B of the genus Nepovirus.     

Delineation of Regions of the Yersinia YopM Protein Required for Interaction with the RSK1 and PRK2 Host Kinases and Their Requirement for Interleukin-10 Production and Virulence

The YopM protein of Yersinia sp. is a type III secreted effector that is required for virulence in murine models of infection. YopM has previously been shown to contain leucine-rich repeats (LRRs) and to interact with two host kinases, RSK1 and PRK2, although the consequence of these interactions is unknown. A series of YopM proteins missing different numbers of LRRs or a C-terminal domain were produced and used for in vitro binding reactions to map domains required for interaction with RSK1 and PRK2. A C-terminal domain of YopM (from LRR12 to the C terminus) was shown to be required for interaction with RSK1, while an internal portion encompassing LRR6 to LRR15 was shown to be required for interaction with PRK2. The virulence of a Yersinia pseudotuberculosis ΔyopM mutant in mice via an intravenous route of infection was significantly attenuated. At day 4 postinfection, there were significantly increased levels of gamma interferon and reduced levels of interleukin-18 (IL-18) and IL-10 in the serum of the ΔyopM-infected mice compared to that of mice infected with the wild type, suggesting that YopM action alters the balance of these key cytokines to promote virulence. The PRK2 and RSK1 interaction domains of YopM were both required for IL-10 induction in vivo, irrespective of splenic colonization levels. In an orogastric model of Y. pseudotuberculosis infection, a ΔyopM mutant was defective in dissemination from the intestine to the spleen and significantly reduced in virulence. In addition, Y. pseudotuberculosis mutants expressing YopM proteins unable to interact with either RSK1 (YopMΔ12-C) or PRK2 (YopMΔ6-15) were defective for virulence in this assay, indicating that both interaction domains are important for YopM to promote pathogenesis.Yersinia pseudotuberculosis is one of three species of Yersinia that are pathogenic for humans. Like Yersinia enterocolitica, it typically causes a self-limiting gastroenteritis, although in rare cases, fatal septicemia may occur. In addition to these two species, Yersinia pestis is a recently evolved species that is the causative agent of plague. Yersinia pathogenesis is generally associated with the presence of a virulence plasmid, pYV (pCD1 in Y. pestis) that encodes a type III secretion system (T3SS) and a repertoire of T3SS effector proteins termed Yops.The Yop proteins have been intensively studied, and the biochemical functions of most are known. The YopE protein is a Rho GTPase-activating protein with specificity for RhoA and Rac1 (6). YopT is a cysteine protease that cleaves both RhoA and Rac1, thereby preventing these proteins from remaining in a membrane-associated location (37). The YpkA protein is unusual in that it consists of two domains; the first is a serine/threonine protein kinase that phosphorylates Gαq, and the second is a C-terminal Rho GDP dissociation inhibitor that is able to inhibit the activity of RhoA and Rac1 (19, 32, 36). YopH is a potent tyrosine phosphatase that dephosphorylates multiple targets, including Fyn-binding protein and p130Cas, which are proteins involved in phagocytosis and cell signaling (5, 21). These four proteins (YopE, YopT, YpkA, and YopH) contribute to the antiphagocytic activity that Yersinia displays toward macrophages and neutrophils (48).In addition, Yersinia species also secrete the YopJ acetyltransferase (YopP in Y. enterocolitica), which acetylates the normally phosphorylatable serine/threonine residues in the activation loop of mitogen-activated protein kinase (MAPK) kinase proteins, as well as in IKKβ (30, 31). This activity inhibits both MAPK and NF-κB signaling, leading to altered cytokine secretion and induction of apoptosis in infected macrophages (34, 50).Another secreted effector protein of Yersinia spp. is YopM, the function of which remains enigmatic. The tertiary structure of Y. pestis YopM has been determined and consists of an N-terminal secretion signal followed by two α helices that serve to initiate the folding of the leucine-rich repeat (LRR) region that makes up the majority of the protein (17). Depending upon the Yersinia species examined, the YopM protein contains 13 to 21 LRRs. There is also a short, unstructured C-terminal domain that is highly conserved among all YopM isoforms. The protein itself has a twisted horseshoe-like structure in which the inside face of the horseshoe is believed to present a binding surface for eukaryotic proteins, as has been proposed for other LRR family proteins (26, 46).While all other Yops have been shown to have enzymatic activity, the YopM protein is believed to be devoid of catalytic activity. Interestingly, YopM has been demonstrated by several groups to localize to the nuclei of both yeast and mammalian cells (4, 40, 41). In Saccharomyces cerevisiae, protein localization seems to depend on the vesicular trafficking system and both the N-terminal half and the C-terminal half of YopM were able to traffic to the nucleus, suggesting that the protein has more than one nuclear localization signal (41). In agreement with this, another group has reported that the C-terminal unstructured domain of YopM and the LRR1-to-LRR3 domain of the protein are able to independently target to the nuclei of both yeast and HEK293T cells (4). Although these studies have produced a general view of the importance of YopM nuclear localization, the consequence of this localization for virulence remains enigmatic.Studies on the role of YopM in Y. pestis pathogenesis using mouse infection models have demonstrated a pronounced loss of virulence of yopM mutants. For example, whereas the wild-type KIM5 strain delivered intravenously had a 50% lethal dose (LD₅₀) of 29 to 42 CFU, the yopM mutant had an LD₅₀ of 3.4 × 10⁵ to 9.8 × 10⁵ CFU (22, 27). Results of studies in which several mutant versions of YopM were constructed suggested that some of the central LRRs are required for function, although the reason for the loss of virulence observed was thought to be an interaction with α-thrombin (22). The importance of YopM/α-thrombin interaction was refuted in a later publication (33). Somewhat paradoxically and in contrast to the first two publications cited, this study suggested that the deletion of yopM from Y. pestis did not result in a pronounced loss of virulence in an intravenous model of infection. In an orogastric infection study with a Y. enterocolitica O:8 strain, deletion of yopM resulted in complete loss of dissemination to the spleen and the liver by 5 days postinfection (45). However, in an intravenous challenge, the yopM mutant showed only a small but significant reduction in spleen and liver colonization, suggesting that YopM may be especially important for promoting bacterial dissemination. A recent publication has suggested that the YopM protein of Y. pestis CO92 is critical for virulence via the subcutaneous route of infection but is dispensable in a pneumonic model of plague (49).The Y. enterocolitica YopM protein has been shown to interact with two eukaryotic protein kinases, RSK1 and PRK2 (29). This interaction increases RSK1 kinase activity, which in turn activates the phosphorylation-dependent kinase activity of PRK2. The net effect of this interaction is increased kinase activity of these proteins toward a heterologous protein substrate. In addition, the Y. enterocolitica protein was shown to modify the phosphorylation pattern of RSK1 in Yersinia-infected J774A.1 cells. This has resulted in a model of YopM function in which these kinases would increase the phosphorylation of substrates of RSK1 and/or PRK2, thereby altering signaling via these proteins (29). The targets of this regulation have not, to date, been identified.Although there has been a great deal of research examining the immune response to Yersinia, until recently there were few clues about what the role of YopM in this response may be. Kerschen et al. demonstrated that loss of yopM from Y. pestis results in increased recruitment of NK1.1+ cells and CD8⁺ T cells to the spleens of infected mice (25). However, a recent publication has suggested that this YopM-mediated NK1.1+ cell depletion is dispensable for virulence, as antibody-mediated ablation of NK1.1+ cells did not rescue the growth limitation of the ΔyopM strain in the spleens or livers of intravenously infected mice (49). Ye et al. presented evidence that neutrophils may be a target of YopM action, since depletion of neutrophils from mice increased the virulence of a Y. pestis yopM mutant in systemic plague (49).In this work, we demonstrate that the LRR6 to LRR15 region (amino acid residues 176 to 379) of YopM is required for PRK2 binding. We also demonstrate that a C-terminal domain of the YopM protein (amino acid residues 299 to 409) is required for binding to RSK1. Deletion of either of these domains from YopM abrogates the virulence of Y. pseudotuberculosis via the orogastric route of infection. In addition, we obtained evidence that YopM''s virulence function is associated with decreased production of gamma interferon (IFN-γ) and increased levels of interleukin-18 (IL-18) and IL-10 in the serum of Y. pseudotuberculosis-infected mice.     

Atg8 and Ire1 in combination regulate the autophagy-related endoplasmic reticulum stress response in Candida albicans

The unfolded protein response (UPR) is an important pathway to prevent endoplasmic reticulum (ER) stress in eukaryotic cells. In Saccharomyces cerevisiae, Ire1 is a key regulatory factor required for HAC1 gene splicing for further production of functional Hac1 and activation of UPR gene expression. Autophagy is another mechanism involved in the attenuation of ER stress by ER-phagy, and Atg8 is a core protein in autophagy. Both autophagy and UPR are critical for ER stress response, but whether they act individually or in combination in Candida albicans is unknown. In this study, we explored the interaction between Ire1 and the autophagy protein Atg8 for the ER stress response by constructing the atg8Δ/Δire1Δ/Δ double mutant in the pathogenic fungus C. albicans. Compared to the single mutants atg8Δ/Δ or ire1Δ/Δ, atg8Δ/Δire1Δ/Δ exhibited much higher sensitivity to various ER stress-inducing agents and more severe attenuation of UPR gene expression under ER stress. Further investigations showed that the double mutant had a defect in ER-phagy, which was associated with attenuated vacuolar fusion under ER stress. This study revealed that Ire1 and Atg8 in combination function in the activation of the UPR and ER-phagy to maintain ER homeostasis under ER stress in C. albicans.