DC-SIGN Neck Domain Is a pH-sensor Controlling Oligomerization: SAXS AND HYDRODYNAMIC STUDIES OF EXTRACELLULAR DOMAIN*

DC-SIGN is a C-type lectin receptor of dendritic cells and is involved in the early stages of numerous infectious diseases. DC-SIGN is organized into a tetramer enabling multivalent interaction with pathogens. Once formed, the DC-SIGN-pathogen complex can be internalized into compartments of increasing acidity. We have studied the pH dependence of the oligomerization state and conformation of the entire extracellular domain and neck region. We present evidence for equilibrium between the monomeric and tetrameric states of the extracellular domain, which exhibits a marked dependence with respect to both pH and ionic strength. Using solution x-ray scattering we have obtained a molecular envelope of the extracellular domain in which a model has been built. Our results highlight the central role of the neck domain in the pH-sensitive control of the oligomerization state, in the extended conformation of the protein, and in carbohydrate recognition domain organization and presentation. This work opens new insight into the molecular mechanism of ligand release and points to new avenues to block the first step of this important infection pathway.Dendritic cells (DCs),² which are found in peripheral tissues and act as sentinels against invading pathogens are considered to be the most efficient professional antigen presenting cells identified so far (1). A major subset of these DCs, dermal DCs, is characterized by the membrane expression of the DC-SIGN (dendritic cell-specific ICAM3 grabbing non-integrin) receptor (CD209). DC-SIGN is a calcium-dependent (C-type) lectin able to recognize highly glycosylated proteins. It is implicated in the early stages of many viral infections (2) and is spatially distributed in well defined membranous microdomains with an average diameter of 200 nm that act as docking platforms for pathogens and endogenous antigen attachment (3, 4). DC-SIGN binds to viral pathogens through their exposed glycoproteins such as HIV-1 envelope protein (gp120) (5), GP1 of Ebola (6), E1 and E2 of HCV (7, 8), and Dengue virus E glycoprotein (9). Apart from the viral world, DC-SIGN has also been implicated in infection processes involving fungi (10, 11), bacteria, such as Neisseria meningitidis (12) and Leishmania pifanoi (13), and parasites, such as Schistosoma mansoni (14). In addition, DC-SIGN mediates DC contacts with endothelial cells, naive T lymphocytes, and neutrophils by interacting with the endogenous adhesion molecules ICAM2 (15), ICAM3 (16), and the CD11b/CD18 integrin (17), respectively.DC-SIGN is a type II membrane protein comprising three main domains: a cytoplasmic region, a transmembrane segment, and an extracellular domain (ECD). The ECD can be divided into two structurally and functionally distinct regions: a neck region involved in the tetramerization of the receptor and a calcium-dependent carbohydrate-recognition domain (CRD), which is at the heart of the molecular recognition processes mediated by DC-SIGN.The cytoplasmic region contains recycling and internalization motifs important for targeting receptors, together with their associated ligands, to subcellular compartments as shown for DEC 205 and DC-SIGN itself (18, 19). Indeed, in some conditions, combined deletion of the triacidic cluster (EEE), the dileucine (LL), and the tyrosine-based (YXXL) internalization motifs induces the loss of DC capacity to enhance T-cell HIV-1 infection (19). The ability of DC-SIGN to promote T-cell infection by HIV-1 involves different pathways depending on the kinetics of the infection process. HIV transfers from DCs to T cells have been described to occur in two phases: a short-term phase (up to 24 h after viral exposure) and a long-term phase (from 24 to 72 h) (20, 21). The long-term phase is proposed to involve membrane fusion and subsequent DC infection and does not need to concern us further in this study. In contrast, short-term phase transfer involves virion internalization into DCs through vesicles. A pH dependence of HIV transfer has been suggested, based on the abrogation of the trans-enhancement of T-cell infection upon intracellular pH neutralization by concanamycin A (19). Indeed, internalization of HIV particles into low pH non-lysosomal compartments has been described in DC-SIGN-expressing cells (19). At the molecular level, a pH dependence of DC-SIGN binding to gp120 has been reported (22), as well as ligand release from DC-SIGN in pH conditions mimicking the acidic luminal environment of endosomes/lysosomes (23). However, no molecular explanation has yet been proposed for this phenomenon that might play a key role in the initiation mechanisms of DC-SIGN-mediated infectious processes. An effect of pH on the CRD binding properties has been reported (22) but no detailed molecular study of the pH dependence of the oligomerization properties of the neck domain, essential for the integrity of ECD, has yet been reported.The neck region comprises seven complete and one incomplete 23-amino acid long repeat. These repeats exhibit a distinct pattern of hydrophobic residues, positioned at regular intervals along an α-helical stretch that could favor coiled-coil interaction between helices from the four chains within a tetramer (24, 25). Comparison of circular dichroism spectra from the entire ECD and the CRD fragment led to a similar suggestion (26). The neck domain, which is responsible for tetramer formation and stability of the whole ECD, is thereby at the onset of the avidity mechanism that endows the DC-SIGN receptor with a very high affinity toward its target as compared with the weak affinity of isolated CRD. Indeed, the dissociation constant of the complex with the HIV gp120 envelope protein is in the nanomolar range with the whole ECD, whereas it is in the millimolar range when using monomeric CRD (22). Moreover, DC-SIGN neck region variants, resulting from alternative splicing and genomic polymorphism, exhibit altered sugar binding (27). DC-SIGN neck polymorphism has recently been correlated with differential susceptibility to HIV-1 and other pathogens as a result of modified DC-SIGN multimerization on the cell surface (27). Beyond the avidity mechanism already mentioned, the association of the four chains within the neck largely constraints the CRD spatial arrangement that must bear on the ligand interaction and recognition properties. However, despite the importance of the DC-SIGN tetrameric state in molecular recognitions, structural information is scarce and mostly restricted to the CRD moiety. High-resolution structures of the CRD in complex with various carbohydrate ligands are available (28–31). The partial structure of a repeat has been obtained using a truncated dimeric form of the related homolog, DC-SIGNR (29, 32). However, structural information on the tetramer is still lacking although it is a prerequisite to a precise understanding of CRD presentation, pathogen recognition, and to rationalize emerging strategies for the design of DC-SIGN multivalent inhibitors (33–36).Our study of the ECD, and more particularly of the neck domain, addresses two issues. First of all, we investigate the role of the neck domain in the structural organization of DC-SIGN ECD. We analyze its stability and dynamic as a function of pH conditions encountered in the various cell compartments. Our studies involve various constructs (CRD, ECD, and neck region) using several techniques: analytical ultracentrifugation, size exclusion chromatography (SEC), and circular dichroism (CD). We show that physiologically encountered pH variations significantly affect the protein oligomerization and structure. Slightly acidic pH destabilizes the tetramer as well as the CRD structure with possible consequences on the strength of ligand binding, which is strongly dependent on the multiplicity of interactions. In parallel, a molecular envelope of DC-SIGN ECD derived from small angle x-ray scattering (SAXS) measurements in solution highlights the extended organization of DC-SIGN and suggests a tighter arrangement of the four CRDs than hereto proposed.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Fatty Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-dependent Manner

The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.Toll-like receptors (TLRs)³ are one of the key pattern recognition receptor families that play a critical role in inducing innate and adaptive immune responses in mammals by recognizing conserved pathogen-associated molecular pattern of invading microbes. So far, at least thirteen TLRs have been identified in mammalian species (1, 2).Lipopolysaccharide (LPS) from Gram-negative bacteria is the ligand for the TLR4 complex (3), whereas, TLR2 can recognize lipoproteins/lipopeptides of Gram-positive bacteria and mycoplasma (1, 2). LPS forms a complex with LPS-binding protein in serum leading to the conversion of oligomeric micelles of LPS to monomers, which are delivered to CD14. Monomeric LPS is known to bind TLR4/MD-2/CD14 complex (4). Lipid A, which possesses most of the biological activities of LPS, is acylated with hydroxy saturated fatty acids. The 3-hydroxyl groups of these saturated fatty acids are further 3-Ο-acylated by saturated fatty acids. Removal of these Ο-acylated saturated fatty acids from Lipid A not only results in complete loss of endotoxic activity, but also makes Lipid A act as an antagonist against the native Lipid A (5, 6). One or more Lipid As containing unsaturated fatty acids are known to be non-toxic and act as an antagonist against endotoxin (7, 8). In addition, deacylated lipoproteins are unable to activate TLR2 and to induce cytokine expression in monocytes (9). Together, these results suggest that saturated fatty acids acylated on Lipid A or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for TLR4 and TLR2. Indeed, it is suggested that the rapid interaction of bacterial lipopeptides with plasma membrane of macrophages occurs via insertion of their acylated saturated fatty acids as determined by electron energy loss spectroscopy and freeze-fracture techniques (10, 11). TLR2 can form a heterodimer with TLR1 or TLR6, which can discriminate the molecular structure of triacyl or diacyl lipopeptides (12–14). So far there is no evidence that microbial ligands for other TLRs are acylated by saturated fatty acids.Results from our previous studies demonstrated that saturated fatty acids activate TLR4 and polyunsaturated fatty acids (PUFA) inhibit both saturated fatty acid- and LPS-induced activation of TLR4 (15, 16). In addition, the saturated fatty acid lauric acid potentiates, but the n-3 PUFA docosahexaenoic acid (DHA) inhibits lipopeptide (TLR2 agonist)-induced TLR2 activation (17). Together, these results suggest that both TLR2 and TLR4 signaling pathways and target gene expression are reciprocally modulated by saturated and polyunsaturated fatty acids. However, the mechanism for this modulation by fatty acids is not understood.TLR4 is recruited to lipid raft factions after cells are treated with LPS and subsequently induces tumor necrosis factor-α expression in RAW264.7 cells (18). This process occurs in an ROS-dependent manner because inhibition of NADPH oxidase suppresses TLR4 recruitment to lipid rafts (19). Methyl-β-dextrin, a lipid raft inhibitor, significantly inhibits the LPS-induced expression of cytokine (19), suggesting that lipid rafts are essential for TLR4-mediated signal transduction and target gene expression. Lipid rafts are a collection of lipid membrane microdomains characterized by insolubility in non-ionic detergents. Lipid rafts serve as a platform where receptor-mediated signal transduction is initiated (20). Lipid rafts have a special lipid composition that is rich in cholesterol, sphingomyelin, and glycolipids (21). The polar lipids in detergent-resistant membrane contain predominantly saturated fatty acyl residues with underrepresented PUFAs (22–24), suggesting that saturated fatty acyl chains favor lipid raft association. On the other hand, n-3 PUFAs displace signaling proteins from lipid rafts by altering lipid composition, and the displacement leads to the suppression of T-cell receptor-mediated signaling (25). It is now well documented that TLRs form homo- or hetero-oligomers (1, 2). TLR4 homodimerization is the initial step of the receptor activation. Results from our previous studies suggest that the molecular target by which saturated fatty acids and n-3 PUFAs reciprocally modulate TLR4 activation is the receptor complex itself or the event leading to the receptor activation instead of the downstream signaling components (15, 16). Therefore, we determined whether the reciprocal modulation of TLR4 activation is mediated by regulation of the dimerization and recruitment of TLR4 into lipid rafts, and if these processes occur in an ROS-dependent manner.     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Mcl-1 Degradation during Hepatocyte Lipoapoptosis

The mechanisms of free fatty acid-induced lipoapoptosis are incompletely understood. Here we demonstrate that Mcl-1, an anti-apoptotic member of the Bcl-2 family, was rapidly degraded in hepatocytes in response to palmitate and stearate by a proteasome-dependent pathway. Overexpression of a ubiquitin-resistant Mcl-1 mutant in Huh-7 cells attenuated palmitate-mediated Mcl-1 loss and lipoapoptosis; conversely, short hairpin RNA-targeted knockdown of Mcl-1 sensitized these cells to lipoapoptosis. Palmitate-induced Mcl-1 degradation was attenuated by the novel protein kinase C (PKC) inhibitor rottlerin. Of the two human novel PKC isozymes, PKCδ and PKCθ, only activation of PKCθ was observed by phospho-immunoblot analysis. As compared with Jurkat cells, a smaller PKCθ polypeptide and mRNA were expressed in hepatocytes consistent with an alternative splice variant. Short hairpin RNA-mediated knockdown of PKCθ reduced Mcl-1 degradation and lipoapoptosis. Likewise, genetic deletion of Pkcθ also attenuated Mcl-1 degradation and cytotoxicity by palmitate in primary hepatocytes. During treatment with palmitate, rottlerin inhibited phosphorylation of Mcl-1 at Ser¹⁵⁹, a phosphorylation site previously implicated in Mcl-1 turnover. Consistent with these results, an Mcl-1 S159A mutant was resistant to degradation and improved cell survival during palmitate treatment. Collectively, these results implicate PKCθ-dependent destabilization of Mcl-1 as a mechanism contributing to hepatocyte lipoapoptosis.Current evidence suggests that hepatic steatosis is present in up to 30% of the American population (1). A subset of these individuals develop severe hepatic lipotoxicity, a syndrome referred to as NASH² (2), which can progress to cirrhosis and its chronic sequela (3, 4). A major risk factor for hepatic lipotoxicity is insulin resistance (5–7), resulting in excessive lipolysis within peripheral adipose tissue with release of high levels of free fatty acids (FFA) to the circulation. Circulating FFA are taken up by the liver via fatty acid transporter 5 and CD36 (8–10), and the bulk of hepatic neutral fat is derived from re-esterification of circulating FFA (8). Current concepts indicate that FFA, and not their esterified product (triglyceride), mediate hepatic lipotoxicity (11, 12). Elevated serum FFA correlate with liver disease severity (13–15), and therapies that enhance insulin sensitivity ameliorate hepatic lipotoxicity, in part, by decreasing plasma FFA (16). Hepatic FFA also accumulate in experimental steatohepatitis, further supporting a role for these nutrients in hepatic lipotoxicity (17). Saturated FFA are more strongly implicated in hepatic lipotoxicity than unsaturated FFA (18, 19). Saturated FFA induce hepatocyte apoptosis (20, 21), a cardinal feature of nonalcoholic fatty liver disease (22), and serum biomarkers of apoptosis are useful for identifying hepatic lipotoxicity (23). Thus, FFA-mediated lipotoxicity occurs, in part, by apoptosis.Apoptosis is regulated by members of the Bcl-2 protein family (24). These proteins can be categorized into three subsets as follows: the guardians or anti-apoptotic members of this family, which include Bcl-2, A1, Mcl-1, Bcl-x_L, and Bcl-w; the multidomain executioners or proapoptotic members of this family, which include Bax and Bak; and the messengers or biosensors of cell death, which share only the third Bcl-2 homology domain and are referred to as BH3-only proteins. This last group of proteins includes Bid, Bim, Bmf, Puma, Noxa, Hrk, Bad, and Bik. We have previously reported that cytotoxic FFA induce Bim expression by a FoxO3a-dependent mechanism that contributes, in part, to lipoapoptosis by activating Bax (20, 21). However, Bax activation can be held in check by anti-apoptotic members of the Bcl-2 family suggesting their function may also be dysregulated during FFA-mediated cytotoxicity.Bcl-2 is not expressed in hepatocytes at the protein level (25), whereas Bcl-w and Bfl-1/A1 knock-out mice have no liver phenotype (26–28). However, both potent anti-apoptotic proteins Bcl-x_L and Mcl-1 are expressed by hepatocytes and exhibit a liver phenotype in knock-out mice (29, 30), whereas up-regulation of Mcl-1 renders hepatocytes resistant to apoptosis (31–33). It has also been posited that cellular elimination of Mcl-1 is a critical step in certain proapoptotic cascades (34, 35). Mcl-1 is unique among Bcl-2 proteins in that it has a short half-life, 30–120 min in most cell types, due to the presence of two sequences rich in proline, glutamic acid, serine, and threonine, which target the protein for rapid degradation by the proteasome (36). Proteasomal degradation of Mcl-1 is promoted by ubiquitination, which in turn is regulated by various kinase cascades (36). Despite its potential importance, a role for Mcl-1 in regulating hepatocyte FFA-mediated lipoapoptosis remains unexplored.Given that FFA induce insulin resistance (37), the kinases potentially regulating lipoapoptosis are likely those also identified in insulin resistance syndromes, especially the novel PKC isoforms PKCδ and PKCθ (38). The novel PKC isoforms are activated by diacylglycerol, which rises in the presence of FFA (39–41), and diacylglycerol levels are significantly increased in NASH (42). A role for PKCδ in apoptosis has not been described. PKCθ has recently been shown to be activated by endoplasmic reticulum stress in liver cells (43) and lipids in vivo (44, 45). Furthermore, PKCθ has also been implicated in apoptosis of Jurkat cells, neuroblastoma cells, and myeloid leukemia cells (46, 47). However, neither its role in mediating lipoapoptosis nor modulating levels/activity of Bcl-2 proteins has been examined.This study addresses the role of Mcl-1 and PKCθ in FFA-induced lipoapoptosis. We identify a pathway that involves PKCθ-dependent proteasomal degradation of Mcl-1. Using inhibitors of various steps along this pathway, along with Mcl-1 mutants that are resistant to proteasomal degradation or Ser¹⁵⁹ phosphorylation, our studies implicate Mcl-1 degradation via a PKCθ-dependent process as a critical step in lipoapoptosis.     

TLR2 Mediates Gap Junctional Intercellular Communication through Connexin-43 in Intestinal Epithelial Barrier Injury

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser³⁶⁸ to prevent spontaneous chronic colitis in MDR1α-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.The intestinal epithelial cell (IEC)³ barrier provides the front line of mucosal host defense in the intestine. The IEC barrier confers anatomic integrity and immunologic protection of the intestinal mucosal surface. Because the IEC barrier constantly faces diverse populations of lumenal microbes and other potential threats, it must exert a highly defined process of continuous discrimination: excluding harmful antigens while allowing host-beneficial substances to permeate (1, 2). Para- and intercellular transit of molecules is modulated by a complex network of closely arranged tight (TJ) and gap junctions (GJ) between juxtaposed IEC. Gap junctional intercellular communication (GJIC) is an essential, but not well understood, mechanism for cellular and tissue homeostasis that coordinates cell-cell passage of ions and small metabolites (<1 kDa). Thus, GJIC regulates cell proliferation, migration, and differentiation (3). GJ channels are formed by hexameric connexins at the plasma membrane. Cx43 is the major connexin and represents a key target in GJIC regulation (4). It is differentially phosphorylated at a dozen or more residues throughout its life cycle (5–9). Alteration of GJIC caused by changes in Cx43 has been proposed to be involved in the pathophysiology of diverse IEC barrier diseases, including inflammatory bowel diseases, necrotizing enterocolitis, cancer, and enteric infection (10–12). However, immune mediators that allow protective GJIC via Cx43 to sustain IEC barrier function during mucosal damage have not yet been identified.Toll-like receptor 2 (TLR2), a member of the TLR family that is constitutively expressed in IEC (13–15), recognizes conserved molecular patterns associated with both Gram-negative and -positive bacteria (16). We have previously shown that commensal-mediated TLR2 helps to maintain functional TJ barrier integrity of the intestinal epithelial layer. TLR2 enhances transepithelial resistance of the IEC barrier by apical redistribution of ZO-1 via protein kinase Cα/δ (17). Treatment with the TLR2 ligand PCSK protects ZO-1-associated IEC barrier integrity and decreases intestinal permeability in acute colitis (18). Previous studies in other cell types have demonstrated that the second PDZ domain of ZO-1 interacts with the carboxyl terminus of Cx43 (19, 20). ZO-1 binds to Cx43 preferentially during the G₀ phase, enhancing assembly and stabilization of GJIC (21, 22). Like TLR2, Cx43 and ZO-1 reside in caveolin-1-associated lipid raft microdomains (23–25). We therefore hypothesized that the binding between ZO-1 and Cx43 may allow TLR2 to control IEC barrier function by GJIC.In this study, we identified a new physiological mechanism of innate immune host defense in the injured intestine. Our findings indicated that Cx43 serves as an important component of the protective innate immune response of the intestinal epithelium. TLR2-induced GJIC via Cx43 appears to control IEC barrier function and restitution during acute and chronic inflammatory damage, enhancing mucosal homeostasis between commensals and host. UC-associated TLR2 mutant results in impaired GJIC by a proteasomal-dependent increase in Cx43 turnover.     

The PRY/SPRY/B30.2 Domain of Butyrophilin 1A1 (BTN1A1) Binds to Xanthine Oxidoreductase: IMPLICATIONS FOR THE FUNCTION OF BTN1A1 IN THE MAMMARY GLAND AND OTHER TISSUES*

Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH- and salt-sensitive manner (K_D = 101 ± 31 nm in 10 mm HEPES, 150 mm NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/SPRY/B30.2 domain. The potential significance of BTN1A1/XOR interactions in the mammary gland and other tissues is discussed.Members of the butyrophilin (BTN)³ gene family are attracting increasing attention because they may play multifunctional roles in diverse physiologies, including lactation (1, 2), selection and regulation of T-cells in the immune system (3–6), and modulation of autoimmune disease (7–9). BTN proteins have the canonical structures of cell surface receptors, which, after an N-terminal signal sequence, generally comprise two exoplasmic Ig folds (10, 11), a membrane anchor and a cytoplasmic domain consisting of a stem region, a PRY/SPRY/B30.2 domain (12, 13), and a cytoplasmic tail at the C terminus (14).The eponymous BTN1A1 protein has been linked to the secretion of milk lipid droplets because it is highly expressed in the mammary epithelium during lactation and is incorporated into the surface membrane coat surrounding cytoplasmic lipid droplets (the milk fat globule membrane (MFGM)) as they bud into milk from the apical surface (15). Furthermore, ablation of the Btn1a1 gene disrupts lipid secretion, causing the accumulation of large pools of triacylglycerol in the cytoplasm of Btn1a1 null mice (1). In a different context, dietary exposure to BTN1A1 in dairy products has been associated with modulation of the autoimmune disease multiple sclerosis because of structural similarities between the IgI fold of BTN1A1 (16) and the IgV fold of myelin oligodendrocyte glycoprotein (MOG) (17) an antigen on the myelin nerve sheath that is a target for autoantibodies in multiple sclerosis patients (8–10).Potential interactions between the exoplasmic Ig folds of several BTN proteins, and putative receptors on immune cells are postulated to regulate positive selection of epidermal γδ-T cells in the case of Skint1 (6) and suppress T-cell activation in the case of BTNL2 (4, 5). In addition, BTN2A1 binds to the C-type lectin, DC-SIGN, on immature dendritic cells (18), and proteins in the BTN3A1–3 subfamily bind to an unidentified ligand on various immune cells (19).Interactions between the cytoplasmic domain of BTN and intracellular proteins have not been investigated in any detail. The intracellular region of most BTNs is dominated by the B30.2 or the PRY/SPRY domain, which comprises two sheets of antiparallel β-strands folded into a β- sandwich, which in some proteins is contiguous at the N terminus with one or two α-helices (20–24) (for a discussion of the relationship between PRY, SPRY, and B30.2 domains, see Ref. (25)). This domain (here abbreviated as B30.2),⁴ is postulated to serve as a protein-binding module, by which proteins interact through the extended surface loops that adjoin individual β-strands (22).One protein that may bind to the cytoplasmic region of BTN proteins (and the B30.2 domain) is the redox enzyme, xanthine oxidoreductase (XOR),⁵ because it was shown to bind to the cytoplasmic domain of mouse BTN1A1 in an in vitro binding assay (26). Furthermore, one XOR-deficient mouse strain (Xdh^+/−) (27) displayed a lactation phenotype similar to that of Btn1a1 null mice (1), suggesting that the two proteins may be functionally linked by direct interaction. These conclusions, however, have been challenged, because XOR does not co-localize with BTN1A1 in immunolabeled freeze-fractured replicates of secreted milk lipid droplets (28), and a second mouse strain deficient in XOR does not appear to have an altered lactation phenotype (29).In this paper, we devise in vivo and in vitro assays to show that the cytoplasmic domain of BTN1A1 binds to XOR via the B30.2 domain and that BTN1A1 is required for the stable association of XOR with the MFGM in vivo. Furthermore, interaction with XOR appears to be limited to BTN1A1 orthologs. These results are discussed in the context of potential functions of BTN1A1 in the mammary gland and other tissues and the relationship of BTN1A1 to other BTN family members.