MHC class I-related antigen-processing machinery component defects in feline mammary carcinoma

Defects in HLA class I antigen-processing machinery (APM) component expression and/or function are frequent in human tumors. These defects may provide tumor cells with a mechanism to escape from recognition and destruction by HLA class I antigen-restricted, tumor antigen-specific cytotoxic T cells. However, expression and functional properties of MHC class I antigens and APM components in malignant cells in other animal species have been investigated to a limited extent. However, this information can contribute to our understanding of the mechanisms underlying the association of MHC class I antigen and APM component defects with malignant transformation of cells and to identify animal models to validate targeted therapies to correct these defects. To overcome this limitation in the present study, we have investigated the expression of the catalytic subunits of proteasome (Y, X, and Z) and of immunoproteasome (LMP2, LMP7, and LMP10) as well as of MHC class I heavy chain (HC) in 25 primary feline mammary carcinomas (FMCs) and in 23 matched healthy mammary tissues. We found a reduced expression of MHC class I HC and of LMP2 and LMP7 in tumors compared with normal tissues. Concordantly, proteasomal cleavage specificities in extracts from FMCs were different from those in healthy tissues. In addition, correlation analysis showed that LMP2 and LMP7 were concordantly expressed in FMCs, and their expression was significantly correlated with that of MHC class I HC. The abnormalities we have found in the APM in FMCs may cause a defective processing of some tumor antigens.     

Tubulin as a molecular component of coated vesicles

Two proteins of 53,000 and 56,000 mol wt have been found to be associated with coated vesicles (CV) purified from bovine brain and chicken liver. These proteins share molecular weights, isoelectric points, and antigenic determinants with alpha- and beta-tubulins purified from bovine brain. Based on SDS PAGE and electron microscopic analysis of controlled pore glass bead exclusion column fractions, both the tubulins and the major CV polypeptide clathrin were found to chromatograph as components of a single kinetic particle. In addition, tubulin and CV antigens assayed by a sensitive enzyme-linked-immunoadsorbent method eluted from the columns with constant stoichiometry. These data provide evidence that tubulin is a molecular component of coated vesicles.     

Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.     

Aftiphilin is a component of the clathrin machinery in neurons

Aftiphilin was identified through a database search for proteins containing binding motifs for the gamma-ear domain of clathrin adaptor protein 1 (AP-1). Here, we demonstrate that aftiphilin is expressed predominantly in brain where it is enriched on clathrin-coated vesicles. In addition to eight gamma-ear-binding motifs, aftiphilin contains two WXXF-acidic motifs that mediate binding to the alpha-ear of clathrin adaptor protein 2 (AP-2) and three FXXFXXF/L motifs that mediate binding to the alpha- and beta2-ear. We demonstrate that aftiphilin uses these motifs for interactions with AP-1 and AP-2 and that it immunoprecipitates these APs but not AP-3 or AP-4 from brain extracts. Aftiphilin demonstrates a brefeldin A sensitive localization to the trans-Golgi network in hippocampal neurons where it co-localizes with AP-1. Aftiphilin is also found at synapses where it co-localizes with synaptophysin and AP-2. Our data suggest a role for aftiphilin in clathrin-mediated trafficking in neurons.     

shutdown is a component of the Drosophila piRNA biogenesis machinery

In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.     

Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery

The roles of the components of the Sec34p protein complex in intracellular membrane trafficking, first identified in the yeast Saccharomyces cerevisiae, have yet to be characterized in higher eukaryotes. We cloned a human cDNA whose predicted amino acid sequence showed 41% similarity to yeast Sec34p with homology throughout the entire coding region. Affinity-purified antibodies raised against the human SEC34 protein (hSec34p) recognized a cellular protein of 94 kDa in both soluble and membrane fractions. Like yeast Sec34p, cytosolic hSec34p migrated with an apparent molecular mass of 300 kDa on a glycerol velocity gradient, suggesting that it is part of a protein complex. Immunofluorescence microscopy localized hSec34p to the Golgi compartment in cells of all species examined, where it co-localized well with the cis/medial Golgi marker membrin and partially co-localized with cis-Golgi network marker p115 and trans-Golgi marker TGN38. The co-localization with membrin was maintained at 15 degrees C and after microtubule depolymerization with nocodazole. During transport of the tsO45 vesicular stomatitis virus G protein through the Golgi, there was significant overlap with the hSec34p compartment. Green fluorescent protein-hSec34 expressed in HeLa cells was restricted to Golgi cisternae, and its membrane association was sensitive to brefeldin A treatment. Taken together, our findings indicate that hSec34p is part of a peripheral membrane protein complex localized on cis/medial Golgi cisternae where it may participate in tethering intra-Golgi transport vesicles.     

EBV protein BNLF2a exploits host tail-anchored protein integration machinery to inhibit TAP

EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host.     

Autophagy in yeast: a review of the molecular machinery

Autophagy is a membrane trafficking mechanism that delivers cytoplasmic cargo to the vacuole/lysosome for degradation and recycling. In addition to non-specific bulk cytosol, selective cargoes, such as peroxisomes, are sorted for autophagic transport under specific physiological conditions. In a nutrient-rich growth environment, many of the autophagic components are recruited for executing a biosynthetic trafficking process, the cytoplasm to vacuole targeting (Cvt) pathway, that transports the resident hydrolases aminopeptidase I and alpha-mannosidase to the vacuole in Saccharomyces cerevisiae. Recent studies have identified pathway-specific components that are necessary to divert a protein kinase and a lipid kinase complex to regulate the conversion between the Cvt pathway and autophagy. Downstream of these proteins, the general machinery for transport vesicle formation involves two novel conjugation systems and a putative membrane protein complex. Completed vesicles are targeted to, and fuse with, the vacuole under the control of machinery shared with other vacuolar trafficking pathways. Inside the vacuole, a potential lipase and several proteases are responsible for the final steps of vesicle breakdown, precursor enzyme processing and substrate turnover. In this review, we discuss the most recent developments in yeast autophagy and point out the challenges we face in the future.     

PBP1 is a component of the Bacillus subtilis cell division machinery

Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell division. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell division proteins. This study proves that PBP1 forms part of the B. subtilis cell division machinery.     

hFis1, a novel component of the mammalian mitochondrial fission machinery

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.     

Multiple residues in the transmembrane helix and connecting peptide of mouse tapasin stabilize the transporter associated with the antigen-processing TAP2 subunit

The type I endoplasmic reticulum (ER) glycoprotein tapasin (Tpn) is essential for loading of major histocompatibility complex class I (MHC-I) molecules with an optimal spectrum of antigenic peptides and for stable expression of the heterodimeric, polytopic TAP peptide transporter. In a detailed mutational analysis, the transmembrane domain (TMD) and ER-luminal connecting peptide (CP) of mouse Tpn were analyzed for their capacity to stabilize the TAP2 subunit. Replacement of the TMD of Tpn by TMDs from calnexin or the Tpn-related protein, respectively, completely abolished TAP2 stabilization after transfection of Tpn-deficient cells, whereas TMDs derived from distantly related Tpn molecules (chicken and fish) were functional. A detailed mutational analysis of the TMD and adjacent residues in the ER-luminal CP of mouse Tpn was performed to elucidate amino acids that control the stabilization of TAP2. Single amino acid substitutions, including a conserved Lys residue in the center of the putative TMD, did not affect TAP2 expression levels. Mutation of this Lys plus four additional residues, predicted to be neighbors in an assumed alpha-helical TMD arrangement, abrogated the TAP2-stabilizing capacity of Tpn. In the presence of a wild-type TMD, also the substitution of a highly conserved Glu residue in the CP of Tpn strongly affected TAP2 stabilization. Defective TAP2 stabilization resulted in impaired cell surface expression of MHC-I molecules. This study thus defines a novel, spatially arranged motif in the TMD of Tpn essential for stable expression of the TAP2 protein and a novel protein interaction mode involving an ER-luminal Glu residue close to the membrane.     

Studies on the physicochemical properties of the new preparation deltaferon as a component of a molecular construct

A molecular construct of a spherical shape containing double-stranded RNA (dsRNA) in the center, coated by a spermidine-polyglucine coat, and carrying recombinant deltaferon—an analogue of recombinant interferon-gamma—has been designed. It is shown that deltaferon in this construct retains its specific activity at a level of 9 × 10⁴ U/mg. It was found that deltaferon and dsRNA in the construct were characterized by enhanced resistance to proteases (trypsin) and nucleases (E. coli ribonuclease) as compared to free deltaferon and dsRNA. An increased resistance of deltaferon in the construct to environmental temperature during storage was also demonstrated.     

Complex protein interactions within the human polyadenylation machinery identify a novel component

Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery. We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the polyadenylation machinery.     

SprB is a cell surface component of the Flavobacterium johnsoniae gliding motility machinery

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces by an unknown mechanism. Transposon insertions in sprB resulted in cells that were defective in gliding. SprB is a highly repetitive 669-kDa cell surface protein, and antibodies against SprB inhibited the motility of wild-type cells. Polystyrene microspheres coated with antibodies against SprB attached to and were rapidly propelled along the cell surface, suggesting that SprB is one of the outermost components of the motility machinery. The movement of SprB along the cell surface supports a model of gliding motility in which motors anchored to the cell wall rapidly propel cell surface adhesins.     

Novel adjuvant based on a proteoliposome-derived cochleate structure containing native lipopolysaccharide as a pathogen-associated molecular pattern

Proteoliposomes (PL) from Neisseria meningitidis B have been widely used as a core antigen for antimeningococcal vaccination. PL contain major outer membrane proteins, LPS and phospholipids, and they induce a strong Th1 immune response, but they have low stability in solution. Attending to the need for new vaccine adjuvants, we developed a highly stable cochleate structure (CS) from PL using a technology that allows easy incorporation of new antigens. We explored the ability of PLCS to activate the immune system and its possible application as an adjuvant for parenteral and mucosal routes. Our results showed that PLCS were able to upregulate the expression of MHC class II and costimulatory molecules on human dendritic cells, as well as being able to stimulate the production of soluble mediators of a Th1 response, such as IL-12 and nitric oxide. High levels of anti-PL IgG were detected in serum after i.m. or mucosal (oral and nasal) administration, but also anti-PL secretory IgA was produced in saliva following nasal delivery. The immune response polarization to a Th1 pattern was confirmed by the induction of IgG2a antibodies, positive delayed type hypersensitivity reactions, and IFN-gamma production by splenocytes from immunized mice. The adjuvant potential was explored using PLCS containing ovalbumin (Ova). PLCS-Ova was able to elicit a substantial increase in anti-Ova IgG compared with Ova alone. In addition, a significant reduction in lesion size was observed in mice immunized with Leishmania major antigens in PLCS after challenge with virulent protozoa, suggesting at least partial modulation of the Th2 environment induced by this parasite. In conclusion, our results support the use of PLCS as a potent Th1 adjuvant for parenteral and mucosal vaccines.     

Immune selection of hot-spot beta 2-microglobulin gene mutations, HLA-A2 allospecificity loss, and antigen-processing machinery component down-regulation in melanoma cells derived from recurrent metastases following immunotherapy

Scanty information is available about the mechanisms underlying HLA class I Ag abnormalities in malignant cells exposed to strong T cell-mediated selective pressure. In this study, we have characterized the molecular defects underlying HLA class I Ag loss in five melanoma cell lines derived from recurrent metastases following initial clinical responses to T cell-based immunotherapy. Point mutations in the translation initiation codon (ATG-->ATA) and in codon 31 (TCA-->TGA) of the beta(2)-microglobulin (beta(2)m) gene were identified in the melanoma cell lines 1074MEL and 1174MEL, respectively. A hot-spot CT dinucleotide deletion within codon 13-15 was found in the melanoma cell lines 1106MEL, 1180MEL, and 1259MEL. Reconstitution of beta(2)m expression restored HLA class I Ag expression in the five melanoma cell lines; however, the HLA-A and HLA-B,-C gene products were differentially expressed by 1074MEL, 1106MEL, and 1259MEL cells. In addition, in 1259MEL cells, the Ag-processing machinery components calnexin, calreticulin, and low m.w. polypeptide 10 are down-regulated, and HLA-A2 Ags are selectively lost because of a single cytosine deletion in the HLA-A2 gene exon 4. Our results in conjunction with those in the literature suggest the emergence of a preferential beta(2)m gene mutation in melanoma cells following strong T cell-mediated immune selection. Furthermore, the presence of multiple HLA class I Ag defects within a tumor cell population may reflect the accumulation of multiple escape mechanisms developed by melanoma cells to avoid distinct sequential T cell-mediated selective events.     

UNCovering the molecular machinery of dependence receptor signaling

Dependence receptors send opposite signals in the presence or absence of ligand, but the underlying mechanisms have been elusive. In this issue of Molecular Cell, Guenebeaud et al. (2010) elucidate the molecular signaling machinery of the dependence receptor UNC5B.     

SecA, an essential component of the secretory machinery of Escherichia coli, exists as homodimer.

Size exclusion chromatography of the cytosolic fraction of SecA-overproducing cells of Escherichia coli suggested that SecA, an essential component of the secretory machinery, exists as an oligomer. The subunit structure of SecA was then studied using a purified specimen. Estimation of the molecular mass by means of ultracentrifugation and chemical crosslinking analysis revealed that SecA exists as a homodimer. The purified SecA was denatured in 6 M guanidine-HCl and renatured to a dimer, which was fully active in terms of translocation, even in the presence of 1 mM dithiothreitol. It is suggested that the dimeric structure is not critically maintained by disulfide bonding between the two subunits, each of which contains four cysteine residues.