FPRP, a major, highly stoichiometric, highly specific CD81- and CD9-associated protein

CD81 and CD9, members of the transmembrane-4 superfamily (TM4SF; tetraspanins), form extensive complexes with other TM4SF proteins, integrins, and other proteins, especially in mild detergents. In moderately stringent Brij 96 lysis conditions, CD81 and CD9 complexes are virtually identical to each other, but clearly distinct from other TM4SF complexes. One of the most prominent proteins within CD81 and CD9 complexes is identified here as FPRP, the 133-kDa prostaglandin F(2alpha) receptor regulatory protein. FPRP, a cell-surface Ig superfamily protein, associates specifically with CD81 or with CD81 and CD9, but not with integrins or other TM4SF proteins. In contrast to other CD81- and CD9-associating proteins, FPRP associates at very high stoichiometry, with essentially 100% of cell-surface FPRP on 293 cells being CD81- and CD9-associated. Also, CD81.CD9.FPRP complexes have a discrete size (<4 x 10(6) Da) as measured by gel permeation chromatography and remain intact after disruption of cholesterol-rich membrane microdomains by methyl-beta-cyclodextrin. Although CD81 associated with both alpha(3) integrin and FPRP in 293 cells, the alpha(3)beta(1).CD81 and CD81.CD9.FPRP complexes were distinct, as determined by immunoprecipitation and immunodepletion experiments. In conclusion, our data affirm the existence of distinct TM4SF complexes with unique compositions and specifically characterize FPRP as the most robust, highly stoichiometric CD81- and/or CD9-associated protein yet described.     

Mitochondrial interferon-induced transmembrane protein-1 is a critical regulator of cell death in MPRO cells

Interferon-induced transmembrane protein 1 (IFITM1) is a transmembrane protein that is an essential mediator of interferon-induced anti-proliferation. Identifying the functional role of IFITM1 has been difficult because of high expression of the protein induces cell death. In this study, we showed that IFITM1 is localized to the mitochondria and that its expression is related to mitochondrial reactive oxygen species (mROS). Overexpression of IFITM1 resulted in a wrinkled and ruptured cell morphology, and subcellular fractionation revealed IFITM1 expression in the mitochondria and the plasma membrane. Interestingly, IFITM1 was expressed at high levels in the mitochondria of MPRO cells, but only at low levels in the plasma membrane. A mitochondrial staining assay confirmed that IFITM1 was localized to the mitochondria in MPRO cells. In addition, IFITM1 expression was significantly increased by treatment with H₂O₂, arachidonic acid, and dithiothreitol. These findings suggest that IFITM1 expression is not restricted to the membrane, but is present in the mitochondria as well. Furthermore, mitochondrial IFITM1 expression is regulated by mROS.     

The CD225 Domain of IFITM3 Is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication

The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3''s distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3''s restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual''s risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.     

Structure of human peptidyl‐prolyl cis–trans isomerase FKBP22 containing two EF‐hand motifs

The FK506‐binding protein (FKBP) family consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. It is assumed that all members are peptidyl‐prolyl cis–trans isomerases with the enzymatic function attributed to the FKBP domain. Six members of this family localize to the mammalian endoplasmic reticulum (ER). Four of them, FKBP22 (encoded by the FKBP14 gene), FKBP23 (FKBP7), FKBP60 (FKBP9), and FKBP65 (FKBP10), are unique among all FKBPs as they contain the EF‐hand motifs. Little is known about the biological roles of these proteins, but emerging genetics studies are attracting great interest to the ER resident FKBPs, as mutations in genes encoding FKBP10 and FKBP14 were shown to cause a variety of matrix disorders. Although the structural organization of the FKBP‐type domain as well as of the EF‐hand motif has been known for a while, it is difficult to conclude how these structures are combined and how it affects the protein functionality. We have determined a unique 1.9 Å resolution crystal structure for human FKBP22, which can serve as a prototype for other EF hand‐containing FKBPs. The EF‐hand motifs of two FKBP22 molecules form a dimeric complex with an elongated and predominantly hydrophobic cavity that can potentially be occupied by an aliphatic ligand. The FKBP‐type domains are separated by a cleft and their putative active sites can catalyze isomerazation of two bonds within a polypeptide chain in extended conformation. These structural results are of prime interest for understanding biological functions of ER resident FKBPs containing EF‐hand motifs.     

The Interferon-induced Transmembrane Proteins,IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry

The interferon-induced transmembrane (IFITM) family of proteins have recently been identified as important host effector molecules of the type I interferon response against viruses. IFITM1 has been identified as a potent antiviral effector against hepatitis C virus (HCV), whereas the related family members IFITM2 and IFITM3 have been described to have antiviral effects against a broad range of RNA viruses. Here, we demonstrate that IFITM2 and IFITM3 play an integral role in the interferon response against HCV and act at the level of late entry stages of HCV infection. We have established that in hepatocytes, IFITM2 and IFITM3 localize to the late and early endosomes, respectively, as well as the lysosome. Furthermore, we have demonstrated that S-palmitoylation of all three IFITM proteins is essential for anti-HCV activity, whereas the conserved tyrosine residue in the N-terminal domain of IFITM2 and IFITM3 plays a significant role in protein localization. However, this tyrosine was found to be dispensable for anti-HCV activity, with mutation of the tyrosine resulting in an IFITM1-like phenotype with the retention of anti-HCV activity and co-localization of IFITM2 and IFITM3 with CD81. In conclusion, we propose that the IFITM proteins act in a coordinated manner to restrict HCV infection by targeting the endocytosed HCV virion for lysosomal degradation and demonstrate that the actions of the IFITM proteins are indeed virus and cell-type specific.     

Mitochondrial localization and pro-apoptotic effects of the interferon-inducible protein ISG12a

ISG12a is one of the most highly induced genes following treatment of cells with type I interferons (IFNs). The encoded protein belongs to a family of poorly characterized, low molecular weight IFN-inducible proteins that includes 6–16 (G1P3), 1–8U (IFITM3), and 1–8D (IFITM2). Our studies demonstrate that the ISG12a protein associates with or inserts into the mitochondrial membrane. Transient expression of ISG12a led to decreased viable cell numbers and enhanced sensitivity to DNA-damage induced apoptosis, effects that were blocked by Bcl-2 co-expression or treatment with a pan-caspase inhibitor. ISG12a enhanced etoposide induced cytochrome c release, Bax activation and loss of mitochondrial membrane potential. siRNA-mediated inhibition of ectopic ISG12a protein expression prevented the sensitization to etoposide-induced apoptosis and also decreased the ability of IFN-β pretreatment to sensitize cells to etoposide, thereby demonstrating a role for ISG12a in this process. These data suggest that ISG12a contributes to IFN-dependent perturbation of normal mitochondrial function, thus adding ISG12a to a growing list of IFN-induced proteins that impact cellular apoptosis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Single Recurrent Mutation in the 5'-UTR of IFITM5 Causes Osteogenesis Imperfecta Type V

Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.-14C>T in the 5'-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.     

Probing the Energetics of Antigen–Antibody Recognition by Titration Microcalorimetry

Our understanding of the energetics that govern antigen–antibody recognition lags behind the increasingly rapid accumulation of structural information on antigen–antibody complexes. Thanks to the development of highly sensitive microcalorimeters, the thermodynamic parameters of antigen–antibody interactions can now be measured with precision and using only nanomole quantities of protein. The method of choice is isothermal titration calorimetry, in which a solution of the antibody (or antigen) is titrated with small aliquots of the antigen (or antibody) and the heat change accompanying the formation of the antigen–antibody complex is measured with a sensitivity as high as 0.1 μcal s⁻¹. The free energy of binding (ΔG), the binding enthalpy (ΔH), and the binding entropy (ΔS) are usually obtained from a single experiment, and no spectroscopic or radioactive label must be introduced into the antigen or antibody. The often large and negative change in heat capacity (ΔC_p) accompanying the formation of an antigen–antibody complex is obtained from ΔHmeasured at different temperatures. The basic theory and the principle of the measurements are reviewed and illustrated by examples. The thermodynamic parameters relate to the dynamic physical forces that govern the association of the freely moving antigen and antibody into a well-structured and unique complex. This information complements the static picture of the antigen–antibody complex that results from X-ray diffraction analysis. Attempts to correlate dynamic and static aspects are discussed briefly.     

A Sorting Signal Suppresses IFITM1 Restriction of Viral Entry

The interferon-induced transmembrane proteins (IFITMs) broadly inhibit virus infections, particularly at the viral entry level. However, despite this shared ability to inhibit fusion, IFITMs differ in the potency and breadth of viruses restricted, an anomaly that is not fully understood. Here, we show that differences in the range of viruses restricted by IFITM1 are regulated by a C-terminal non-canonical dibasic sorting signal KRXX that suppresses restriction of some viruses by governing its intracellular distribution. Replacing the two basic residues with alanine (KR/AA) increased restriction of jaagsiekte sheep retrovirus and 10A1 amphotropic murine leukemia virus. Deconvolution microscopy revealed an altered subcellular distribution for KR/AA, with fewer molecules in LAMP1-positive lysosomes balanced by increased levels in CD63-positive multivesicular bodies, where jaagsiekte sheep retrovirus pseudovirions are colocalized. IFITM1 binds to cellular adaptor protein complex 3 (AP-3), an association that is lost when the dibasic motif is altered. Although knockdown of AP-3 itself decreases some virus entry, expression of parental IFITM1, but not its KR/AA mutant, potentiates inhibition of viral infections in AP-3 knockdown cells. By using the substituted cysteine accessibility method, we provide evidence that IFITM1 adopts more than one membrane topology co-existing in cellular membranes. Because the C-terminal dibasic sorting signal is unique to human IFITM1, our results provide novel insight into understanding the species- and virus-specific antiviral effect of IFITMs.     

The human FK506-binding proteins: characterization of human FKBP19

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas.     

The human CD38 gene: polymorphism, CpG island, and linkage to the CD157 (BST-1) gene

 CD38 is a leukocyte activation antigen and ectoenzyme [NAD(P)⁺ glycohydrolase; EC 3.2.2.6] involved in numerous immune functions. The human CD38 gene is complex [eight exons, >80 kilobases (kb) long] located on Chromosome 4p15, and part of the eukaryotic NAD⁺ glycohydrolase/ADP-ribosyl cyclase gene family. Because of the increasing relevance of the CD38 molecule in the host immune response to infectious, tumoral, and metabolic diseases, we investigated the genetic variability and linkage of the human CD38 locus. We report that (1) the restriction endonuclease Pvu II identifies a bi-allelic polymorphism here defined as formed by the alleles CD38 ^* A (12 kb) and CD38 ^* B (9/2.5 kb); (2) their frequency in the healthy Italian Caucasian population is 14% and 86%, respectively; (3) the polymorphic Pvu II site is located at the 5′ end of the first intron of the CD38 gene; (4) in conjunction with the polymorphic site, we identified a 900 base pair CpG island associated with the CD38 gene, with two potential Sp1 binding sites; (5) the CpG island may play a role in the regulation of CD38 expression and is hypomethylated in various cell lines; (6) by pulsed-field gel electrophoresis we show that CD38 and its paralogue, the bone-marrow stromal cell antigen BST-1 (CD157), map to the same 800 kb Avi II fragment, indicating that the two human ecto-NADase genes are closely linked. Received: 16 December 1998 / Revised: 26 January 1999     

PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins.

CD81 exerts a range of interesting effects on T cells including early thymocyte differentiation, LFA-1 activation, and provision of costimulation. To better understand the mechanisms by which CD81 influences T cell function we evaluated CD81 molecular complexes on T cells. The most prominent CD81-associated cell surface protein on thymocytes as well as a number of T cell and B cell lines has an apparent molecular mass of 75 kDa. The 75-kDa protein was purified and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry followed by postsource-decay profiling. p75 is a novel type I transmembrane protein of the Ig superfamily which is most similar to FPRP. We cloned and sequenced both human and mouse PG regulatory-like protein (PGRL) and characterized mouse PGRL expression in both lymphocytes and nonlymphoid tissues. The discovery of PGRL allows for the clustering of a small family of related proteins including PGRL, FPRP, V7/CD101, and IGSF3. Expression constructs containing various domains of PGRL with an epitope tag were coexpressed with CD81 and used to determine that the interaction of CD81 with PGRL requires the membrane distal Ig3-Ig4 domains of PGRL. Although it remains to be determined whether PGRL possesses PG regulatory functions, transwell chamber experiments show that PGs and CD81 coordinately regulate T cell motility.     

Identification and characterization of a pig FKBP5 gene with a novel expression pattern in lymphocytes and granulocytes

Abstract

The immunophilins are an important group of regulatory molecules in the immune system. FKBP5, expressed throughout mammals and in fish and birds, functions in both physiological and pathogenic pathways, including innate immunity and steroid-based diseases. In this study, we cloned the first porcine FKBP5 from Rongchang pig by the rapid amplification of cDNA ends technique. The full-length cDNA is 4097?bp, with an open reading frame of 1371?bp that codes for a 457-aa protein. Western blotting detected the porcine FKBP5 protein at highest levels in thymus, followed by spleen and lung. Immunohistochemistry detected the porcine FKBP5 protein in lymphocytes and granulocytes of the blood, and flow cytometry identified greater expression in unactivated (vs. activated) T lymphocytes. Finally, the expression level of porcine FKBP5 in the granulocytes was found to decline significantly from the time of birth to one-year-old. These collective data suggest that the newly identified porcine FKBP5 may function in activation of T cells in pig and in innate immunity in the newborn pig in particular.     

A new epoxidic ganoderic acid and other phytoconstituents from Ganoderma lucidum

A new epoxidic ganoderic acid, 8α,9α-epoxy-3,7,11,15,23-pentaoxo-5α-lanosta-26-oic acid (1), together with the known compounds 3β-hydroxy-7,11,15,23-tetraoxo-5α-lanosta-8-en-26-oic acid (2), ergosta-7,22-diene-3β-yl pentadecanoate (3), ergosta-7,22-diene-3β-ol (4), β-sitosterol (5), fatty acids (6–10), fatty acid ester (11) and octadecane (12) were isolated from the fruiting bodies of Ganoderma lucidum from south India. Their structures were determined by ¹H, ¹³C, ¹³C DEPT, ¹H–¹H COSY, HMBC, HSQC, NOESY NMR, FT-IR, UV–vis and FABMS spectral analysis. Compounds (1–3) exhibited good antifungal activity against Candida albicans in disc diffusion assay (100 μg/disc). Steroid ester (3) showed moderate anti-inflammatory activity (59.7% inhibition, 100 mg/kg body weight) in carrageenan-induced paw edema.     

ATRA-induced HL-60 myeloid leukemia cell differentiation depends on the CD38 cytosolic tail needed for membrane localization, but CD38 enzymatic activity is unnecessary

Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11–20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11–20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11–20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function.