Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world's population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered “hypervirulent” as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains.
In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro ؊ Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro ؊ Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis. Viruses in the genusNorovirus of the family Caliciviridae are important etiologic agents of acute gastroenteritis (12). A recent study estimates that as many as 23 million cases of norovirus-related illness occur in the United States each year (28). Noroviruses are often associated with outbreaks in settings that include hospitals, nursing homes, cruise ships, schools, and military operations (9). Studies also have suggested a significant role for these viruses as agents of pediatric gastrointestinal disease (31, 34).Development of control strategies for the noroviruses has been hampered by the unavailability of permissive cell culture systems. However, recent advances in the understanding of norovirus biology and replication have been facilitated by the availability of recombinant proteins expressed from cloned viral genes (15). The approximately 7.6-kb polyadenylated positive-sense RNA genome of norovirus is organized into three open reading frames (ORFs) (16, 21). The first ORF (ORF1) encodes a 200-kDa polyprotein that is processed by the viral 3C-like (3CL) proteinase to release both precursors and cleavage end products (2,13,22,23,40). The second ORF (ORF2) encodes the major structural protein, VP1 (15), and the third ORF (ORF3) encodes the minor structural protein, VP2 (8). Proteolytic cleavage maps of the ORF1 polyprotein have been determined in in vitro studies for strains representing the two major genetic groups of the noroviruses that are associated with human disease, genogroup I (GI) and genogroup II (GII) (2,3,13,23,40). The proposed gene order for the norovirus ORF1 polyprotein is N-terminal protein (Nterm), NTPase, p22 (GI) or p20 (GII), VPg, proteinase (Pro), and polymerase (Pol) (2,22,23,40).The calicivirus RNA-dependent RNA polymerase (RdRp) is relat...
The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a highthroughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases. Keywords Isopeptidases; Protein degradation; N terminus; 3D polymeraseThe ubiquitin-proteasomal pathway regulates the cellular content and/or compartmentalization of many proteins [1,2] and is a promising, albeit underexploited, area for drug discovery. The therapeutic value of this pathway was recently validated by the introduction and clinical success of Velcade, a proteasome inhibitor, for the treatment of refractory relapsed multiple myeloma [3]. Other ubiquitin-like proteins (UBLs) 1 have recently been shown to contribute similarly to the cellular regulation of proteins, with the most extensively characterized UBL being small ubiquitin-like modifying protein (SUMO) [4].Destruction of a targeted protein via the ubiquitin system initially involves recognition by a multienzyme system that attaches ubiquitin to the target protein. Energy is required to activate ubiquitin at its carboxy terminus. Activation is catalyzed by the enzyme E1, which links the ubiquitin C terminus to a cysteine side chain of the enzyme. This activated ubiquitin is * Corresponding author. Fax: +1 610 644 8616., E-mail address: mattern@progenra.com (M.R. Mattern).. 1 Abbreviations used: UBL, ubiquitin-like protein; SUMO, small ubiquitin-like modifying protein; UBP/USP, ubiquitin-specific protease; UCH, ubiquitin terminal hydrolase; DUB, deubiquitinating enzyme; Ub-AMC, Ub-7-amino-4-methylcoumarin; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl-β-D-thiogalactopyranoside; PMSF, phenylmethylsulfonylfluoride; GFP, green fluorescent protein; DTT, dithiothreitol. transferred to a second enzyme of the series, E2 (ubiquitin-conjugating protein), as a thioe...
Phosphatidylinositol-3 kinases (PI3Ks) modulate cellular growth, proliferation, and survival; dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or transmembrane inhibitor of PI3K [TrIP]) is a putative transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular domain with homology to the inter-SH2 domain of the PI3K regulatory subunit p85, but the mechanism of TrIP function is poorly understood. We show that both the kringle and p85-like domains are necessary for TrIP inhibition of PI3K and that TrIP is down-modulated from the surface of T cells during T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by mediating oligomerization. Using an inducible knockout mouse model, we show that TrIP-deficient T cells exhibit more robust activation and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is a negative regulator of T cell activation and may represent a novel target for immune modulation.
Phosphatidylinositol-3 kinases (PI3Ks) modulate numerous cellular functions, including growth, proliferation and survival. Dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or Transmembrane Inhibitor of PI3K - TrIP) is a novel transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular "p85-like" domain with homology to the inter-SH2 domain of the regulatory subunit of PI3K. Although TrIP has been shown to bind to the p110 catalytic subunit of PI3K in fibroblasts, the mechanism by which TrIP functions is poorly understood. We show that both the kringle and "p85-like" domains are necessary for TrIP inhibition of PI3K. We also demonstrate that TrIP protein is down-modulated from the surface of T cells to allow T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by binding an as-yet-unidentified ligand. Using an inducible knockout mouse model that we developed, we show that TrIP-deficient T cells exhibit more robust T cell activation, show a preference for Th1 polarization and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is an important negative regulator of T cell activation and may represent a novel target for immune modulation therapies.
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