Short Cytoplasmic Domain Research Articles

Facilitating and restraining virus infection using cell-attachable soluble viral receptors

SARS-CoV-2 uses the receptor binding domain (RBD) of its spike protein to recognize and infect host cells by binding to the cell surface receptor angiotensin converting enzyme 2 (ACE2). The ACE2 receptor is composed of peptidase domain (PD), collectrin-like domain, transmembrane domain, and short cytoplasmic domain, and may exist as a dimer on cell surface. The RBD binding site is located atop of the ACE2 PD, but the involvement of other domains in virus infection is uncertain. We found that the ACE2 PD alone, whether anchored to cell membrane via a glycosylphosphatidylinositol anchor or attached to another surface protein, is fully functional as a receptor for spike-mediated cell fusion and virus infection. However, for ACE2 to function as the viral receptor, the RBD binding site must be positioned in close proximity to the cell membrane. Elevating the surface height of ACE2 using long and rigid protein spacers reduces or eliminates cell fusion and virus infection. Moreover, we found that the RBD-targeting neutralizing antibodies, nanobodies, and de novo designed miniprotein binders, when present on cell surface, also act as viral receptors, facilitating cell fusion and virus infection. Our data demonstrate that RBD binding and close membrane proximity are essential properties for a receptor to effectively mediate SARS-CoV-2 infection. Importantly, we show that soluble RBD-binders can be engineered to make cells either susceptible or resistant to virus infection, which has significant implications for antiviral therapy and various virus-mediated applications.

NMR Structural Study of Syndecan-4 Transmembrane Domain with Cytoplasmic Region

Syndecan-4 (SDC4) consists of transmembrane heparan sulfate proteoglycan (HSPG) belonging to the syndecan family. It is present in most cell types of Mammalia. Its structure contains a heparan-sulfate-modified extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain. Regarding the overall cellular function of SDC4, other cells or ligands can bind to its ecto-domain. In addition, 4,5-bisphosphate phosphatidylinositol (PIP2) or protein kinase Cα can bind to its cyto-domain to activate downstream signaling pathways. To understand the signal transduction mechanism of syndecan, it is important to know the interactions between their actual structure and function in vivo. Therefore, it is important to identify the structure of SDC4 to understand the ligand binding behavior of SDC4. In this study, expression and purification were performed to reveal structures of the short ecto-domain, the transmembrane domain, and the cytoplasmic domain of Syd4-eTC (SDC4). Solution-state NMR spectroscopy and solid-state NMR spectroscopy were used to study the structure of Syd4-eTC in membrane environments and to demonstrate the interaction between Syd4-eTC and PIP2.

Identification and functional analysis of Mannose receptor in Asian swamp eel (Monopterus albus) in response to bacterial infection

Mannose receptor (MR), as a member of the C-type lectin (CLEC) family, plays an important role in the internalize pathogen-associated ligands and activate immune response. In the present study, MR was identified and characterized from Asian swamp eel (Monopterus albus) (namely MaMR). The open reading frame of MaMR was 4311 bp in length encoding 1437 amino acids of a ∼162.308 kDa protein, including a cysteine-rich (CR) domain, a fibronectin type II (FNII) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. Phylogenetic analysis indicated that MaMR shared the highest similarity with that of Paralichthys olivaceus. The expression of MaMR was found in all the examined tissues, with the highest expression in the spleen and kidney. After injection with Edwardsiella tarda, the transcript level of MaMR was initially reduced and then significantly elevated in the liver, spleen, foregut and hindgut. In the isolated peripheral blood leukocytes, the expression of MaMR was significantly induced post stimulated with LPS and LTA. Then the MaMR-CTLD4-8 recombinant protein was purified. Bacterial agglutination and binding assay showed that rMaMR-CTLD4-8 could bind with both Gram-positive and Gram-negative bacteria and agglutinate bacteria in the presence of calcium in vitro. Further analysis revealed that MaMR and TLR2 coordinately induced the expression of TRAF6 and promoted the phosphorylation level of p65, leading to the expression of proinflammatory cytokines il-1β and tnf-α in EPC cells. Taken together, these results reveal that MaMR plays an important role in the immune response of fish to pathogen infections.

The mucin-like, secretory type-I transmembrane glycoprotein GP900 in the apicomplexan Cryptosporidium parvum is cleaved in the secretory pathway and likely plays a lubrication role

BackgroundCryptosporidium parvum is a zoonotic parasite and member of the phylum Apicomplexa with unique secretory organelles, including a rhoptry, micronemes and dense granules that discharge their contents during parasite invasion. The mucin-like glycoprotein GP900 with a single transmembrane domain is an immunodominant antigen and micronemal protein. It is relocated to the surface of excysted sporozoites and shed to form trails by sporozoites exhibiting gliding motility (gliding sporozoites). However, the biological process underlying its relocation and shedding remains unclear. The primary aim of this study was to determine whether GP900 is present as a transmembrane protein anchored to the plasma membrane on the surface of sporozoites and whether it is cleaved before being shed from the sporozoites.MethodsTwo anti-GP900 antibodies, a mouse monoclonal antibody (mAb) to the long N-terminal domain (GP900-N) and a rabbit polyclonal antibody (pAb) to the short C-terminal domain (GP900-C), were produced for the detection of intact and cleaved GP900 proteins in sporozoites and other parasite developmental stages by microscopic immunofluorescence assay and in discharged molecules by enzyme-linked immunosorbent assay.ResultsBoth anti-GP900 antibodies recognized the apical region of unexcysted and excysted sporozoites. However, anti-GP900-N (but not anti-GP900-C) also stained both the pellicles/surface of excysted sporozoites and the trails of gliding sporozoites. Both antibodies stained the intracellular meronts, both developing and developed, but not the macro- and microgamonts. Additionally, the epitope was recognized by anti-GP900-N (but not anti-GP900-C) and detected in the secretions of excysted sporozoites and intracellular parasites.ConclusionsGP900 is present in sporozoites and intracellular meronts, but absent in sexual stages. It is stored in the micronemes of sporozoites, but enters the secretory pathway during excystation and invasion. The short cytoplasmic domain of GP900 is cleaved in the secretory pathway before it reaches the extracellular space. The molecular features and behavior of GP900 imply that it plays mainly a lubrication role.Graphical

Molecular structure, expression, and the emerging role of Siglec-15 in skeletal biology and cancer.

Siglec-15, a Siglec family member and type-1 transmembrane protein, is expressed mainly in human macrophages and dendritic cells. It is comprised of a lysine-containing transmembrane domain, two extracellular immunoglobulin (Ig)-like domains and a short cytoplasmic domain. Siglec-15 is highly conserved in vertebrates and acts as an immunoreceptor. It exerts diverse functions on osteoclast physiology as well as the tumor microenvironment. Siglec-15 interacts with adapter protein DAP12 - Syk signaling pathway to regulate the RANKL/RANK-mediated PI3K, AKT, and ERK signaling pathways during osteoclast formation in vitro. Consistently, the lack of the Siglec-15 gene in mice leads to impaired osteoclast activity and osteopetrosis in vivo. In addition, Siglec-15 is expressed by tumor-associated macrophages (TAMs) and regulates the tumor microenvironment by activating the SYK/MAPK signaling pathway. Interestingly, Siglec-15 shares sequence homology to programmed death-ligand 1 (PD-L1) and has a potential immune-regulatory role in cancer immunology. Thus, Siglec-15 might also represent an alternative target for the treatment of cancers that do not respond to anti-PD-L1/PD-1 immunotherapy. Understanding the role of Siglec-15 in osteoclastogenesis and the tumor microenvironment will help us to develop new treatments for bone disorders and cancer.

Expression and functional characterization of the mannose receptor (MR) from Nile tilapia (Oreochromis niloticus) in response to bacterial infection

Mannose receptor (MR) as a member of the pattern recognition receptors (PRRs) plays an important role in the immune response. In mammals, the role of MR in the regulation of phagocytosis is clarified; however, its contribution to opsonize phagocytosis remains unclear in bony fish. In this study, the expression pattern of Nile tilapia mannose receptor gene (OnMR) was investigated and its regulation of the phagocytosis of monocytes/macrophages to pathogenic bacteria was identified. The full-length of OnMR open reading frame is 4314 bp, encoding a peptide containing 1437 amino acid residues. The deduced amino acid sequence revealed that OnMR contained a cysteine-rich domain, a fibronectin type II domain, multiple C-type lectin-like domains, a transmembrane domain and a short cytoplasmic domain. Tissue distribution analysis showed the OnMR transcripts was widely distribute in the ten detected tissues, and highly expressed in head kidney, hind kidney, intestine and spleen. After S. agalactiae and A. hydrophila infection, the expression of OnMR in head kidney and spleen increased significantly. Moreover, the expression of OnMR in MO/Mø were also upregulated post the infection of bacteria and mannose solutions in vitro. This suggested that MR, as a mannose receptor on macrophage surface, could respond strongly to the stimulation of pathogenic bacteria. In addition, the (r)OnMR protein could effectively bind and agglutinate S. agalactiae and A. hydrophila, and regulate the phagocytic ability of monocytes/macrophages to pathogenic bacteria. These results suggest that OnMR is involved in response against bacterial infection in Nile tilapia, and this study will help us better understand the function of MR in teleost fish.

Electrophysiological and Biochemical Characterization of the Human P2X7B Receptor Inxenopus Laevisoocytes

P2X receptors (P2XR) are trimeric ATP-gated ion channels consisting of seven isoforms, P2X1-P2X7, which are permeable to cations. A hallmark of the 595 residues long P2X7R subunit is that its 2nd transmembrane domain (TM2) is linked with a cytoplasmic domain that is significantly larger than that of any other P2XR. From the partial or complete loss of the ATP4--activated ion channel activity due to various C-terminal deletions, we know that this endodomain plays an important functional role. Interestingly, several human P2X7 (hP2X7) splice variants were identified, including four C-terminally truncated variants named hP2X7B, hP2XE, hP2XG and hP2X7J. The amino acid sequences of P2X7A and P2X7B are identical up to Leu346, which lies approximately in the middle of TM2. The subsequent hP2X7B-unique part comprises the C-terminal portion of TM2 (347VRDSLFHALG356), followed by a very short cytoplasmic domain of only eight residues in total (357KWFGEGSD364). Here, we compared the P2X7AR and hP2X7BR as ATP4--gated cation channels and their oligomeric structure and expression on the cell surface. Shortening the hP2X7B variant C-terminal to Leu346 to study only the hP2X7A-identical portion reduced surface expression and homotrimeric assembly. On the other hand, if the truncation mutant contained all TM2 residues including Gly356, but without the eight residues of the cytoplasmic tail, homotrimeric assembly and surface expression was as efficient as with the full-length hP2X7B. ATP4--induced currents mediated by hP2X7BR desensitized relatively quickly as compared to the hP2X7AR, which has a non-desensitizing phenotype. The desensitizing behavior of hP2X7BR supports the view that the long C-terminal endodomain of the non-desensitizing hP2X7AR serves as a gating module that by forming a cytoplasmic cap, stabilizes the ATP4--induced open state of the P2X7AR.However, C-terminal endodomain is not absolutely needed for the channel function.

Structural characterization of a dimeric complex between the short cytoplasmic domain of CEACAM1 and the pseudo tetramer of S100A10-Annexin A2 using NMR and molecular dynamics

AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process. Using NMR and molecular dynamics, we show that AIIt and its pseudoheterodimer interacts with two molecules of short cytoplasmic domain isoform peptides, and that interaction depends on the binding motif 454-Phe-Gly-Lys-Thr-457 where Phe-454 binds in a hydrophobic pocket of AIIt, the null mutation Phe454Ala reduces binding by 2.5 fold, and the pseudophosphorylation mutant Thr457Glu reduces binding by three fold. Since these two residues in CEACAM1-SF were also found to play a role in the binding of calmodulin and G-actin at the membrane, we hypothesize a sequential set of three interactions are responsible for regulation of cis- to trans-dimerization of CEACAM1. The hydrophobic binding pocket in AIIt corresponds to a previously identified binding pocket for a peptide found in SMARCA3 and AHNAK, suggesting a conserved functional motif in AIIt allowing multiple proteins to reversibly interact with integral membrane proteins in a calcium dependent manner.

Abstract 4670: TIP60 regulates alternative splicing of Integrin subunit alpha 6

Abstract Differences in RNA processing, specifically RNA splicing, contribute to enormous diversity that can be generated from the eukaryotic genome. Given the increasing evidence to show that splicing can occur co-transcriptionally, chromatin structure and histone modifications have been reported to affect the choice of a splice site. For instance, HDAC inhibitor treatment has been shown to affect alternative splicing. TIP60 is a haploinsufficient tumor suppressor gene that is frequently downregulated in cancers. TIP60 is a lysine acetyltransferase that acetylates histone and non-histone proteins. Since TIP60 is a writer of histone and non-histone modifications, we investigated TIP60's role in modulating alternative splicing. To understand TIP60's role in alternative splicing, RNA sequencing was performed in MCF10A cells that were (a) transiently depleted of TIP60 (b) stably overexpressing siRNA-resistant wild-type TIP60 (MCF10A*WT) and (c) stably overexpressing siRNA-resistant catalytically dead TIP60 (MCF10A*KD). Differential splicing analysis was performed using MISO program. We found 130 genes to be differentially spliced when TIP60 was depleted and 59 genes were differentially spliced in MCF10A*KD compared to MCF10A*WT. For both comparisons, the majority of the differential splicing events were skipped exon events. Upon overlapping the differential skipped exon events, we found significant exon 25 skipping of ITGA6, resulting in an isoform that has a shorter cytoplasmic domain. TIP60 depletion results in an increase in the ITGA6 isoform that lacks exon 25, which has been implicated to promote tumor initiation in breast cancer. To identify the splicing factor that regulates this event, we performed a siRNA screen of over 70 splicing factors and identified ESRP2 to regulate splicing of ITGA6 exon 25. The consequence and biological significance of TIP60-ESRP2 mediated alternative splicing will be discussed at the conference. Citation Format: Shreshtha Sailesh Bhatia, H. Phillip Koeffler, Sudhakar Jha. TIP60 regulates alternative splicing of Integrin subunit alpha 6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4670.

Lysosomal integral membrane protein LGP85 (LIMP-2) is ubiquitinated at the N-terminal cytoplasmic domain

LGP85/LIMP-2 is a type III transmembrane glycoprotein of lysosomes, which traverses the membrane twice with an N-terminal uncleaved signal sequence and C-terminal hydrophobic domain. In addition to functioning as a receptor for a lysosomal enzyme β-glucocerebrosidase and for several enteroviruses, LGP85 plays a key role in the biogenesis and maintenance of endosomal/lysosomal compartments (ELCs). Our previous studies have demonstrated that overexpression of rat LGP85 into COS cells results in the enlarged ELCs, from where membrane trafficking is impaired. We show here that rat LGP85 is polyubiquitinated at the N-terminal short cytoplasmic domain that comprises of only three amino acid residues, alanine, arginine, and cysteine. Replacement of either arginine or cysteine with alanine within the N-terminal cytoplasmic domain did not influence the ubiquitination of LGP85, thereby indicating that ubiquitin (Ub) is conjugated to the α-NH2 group of the N-terminal alanine residue. Furthermore, we were able to define a domain necessary for ubiquitination in a region ranging from the amino acids 156 to 255 within the lumenal domain of LGP85. This is the first report showing that the integral lysosomal membrane protein LGP85 is ubiquitinated.

Limited Protection Conferred by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Spike Protein.

Recombinant Newcastle disease virus (NDV) LaSota (LS) expressing secreted trimeric spike (S)-ectodomain (Se) of infectious bronchitis virus (IBV) (rLS/IBV.Se) was developed and evaluated for protection conferred against IBV challenge. The IBV S-ectodomain protein, which is S excluding the transmembrane anchor and short cytoplasmic domain of S2, expressed from recombinant LS corresponds to an Arkansas (Ark)-type IBV. In a first experiment, chickens were primed at 1 day of age or primed at 1 day of age and boosted at 14 days of age with 104 50% embryo infectious doses (EID50)/bird of rLS/IBV.Se and challenged with a virulent Ark strain. A single vaccination proved completely ineffective at protecting chickens against challenge, whereas priming and boosting reduced clinical signs and tracheal lesions but did not reduce viral load in lachrymal fluids. In experiment 2, the vaccine dose was increased to 107 EID50/bird and a different virulent Ark strain was used for challenge. In addition, chickens were singly immunized on either day 1 or day 10 after hatch. NDV antibody levels detected in vaccinated chickens were moderate, with hemagglutination inhibition titers varying between 4 and 5 log2. Slightly higher antibody levels to NDV were observed in chickens vaccinated on day 10 versus day 1 but without the difference achieving statistical significance. In contrast, antibody responses measured using recombinant IBV S1 protein-coated ELISA plates were significantly greater in chickens vaccinated on day 10 than on day 1. The use of a higher rLS/IBV.Se dose substantially enhanced the success of a single vaccination compared to experiment 1. Signs and tracheal lesions were reduced more effectively in chickens vaccinated at day 10 after hatch. However, as in experiment 1, vaccination did not reduce the viral loads in tear fluids of challenged chickens. Similar results, in which no reduction in viral load in the trachea was apparent from rLS/IBV.S vaccination, have been obtained by others. Further work is needed to understand the immune responses induced by this recombinant virus that seems to provide some protection against the disease but does not reduce viral loads in the upper respiratory tract.

Abstract 4509: Overexpression of CADM1 enhances malignant features of small-cell lung cancer

Abstract Small-cell lung cancer (SCLC) is one of the representative intractable cancers with the 5-year survival of less than 10 %. Novel approaches to the treatment of SCLC are required because recurrence arises inevitably after the initial chemotherapy. CADM1 was originally identified as a tumor suppressor of non-small cell lung cancer (NSCLC) which encodes a member of immunoglobulin superfamily cell adhesion molecules. CADM1 consists of an extracellular domain with three immunoglobulin-like loops, a trans-membrane domain and a short cytoplasmic domain containing a protein 4.1-binding motif (4.1BM) and a PDZ-II-binding motif. We have reported that CADM1 is highly expressed in 90% of human SCLC cell lines and promotes subcutaneous tumor formation of SCLC cells in nude mice. In this study, we performed clinicopathological evaluation of CADM1 in SCLC and analyzed the molecular mechanisms of malignant progression of SCLC by overexpressing CADM1. Firstly, we performed immunohistochemical analysis of CADM1 expression in high-grade neuroendocrine tumor (HGNEC) of the lung, including SCLC and large cell neuroendocrine carcinoma (LCNEC). We found that CADM1 protein was expressed in 75% (33/44) of SCLC tumors and correlated with the nodal involvement in lung HGENC. To understand pathological significance of CADM1 overexpression in SCLC, we introduced a full-length CADM1 and its mutants in the extracellular or intracellular domain into SCLC cells, SBC5, lacking endogenous CADM1 expression and assessed their malignant features in comparison with control SBC5 cells. CADM1 expression significantly enhanced colony formation in soft agar and subcutaneous tumor formation in nude mice of SBC5, whereas a mutant in 4.1BM did not. Then, we screened chemical library of 170 small molecules of known function to identify compounds which inhibit anchorage-independent growth of SCLC cells enhanced by CADM1. Inhibitors of PI3K, Akt and PKA significantly suppressed cell growth of SBC5, suggesting that CADM1 activates PI3K/Akt and PKA signaling pathways through its 4.1BM and promotes anchorage-independent growth of SCLC cells. We also found that orthotopic injection of NCI-N417 cells overexpressing CADM1 into the lung of nude mice significantly enhanced their metastasis to mediastinal lymph nodes. These results suggest that CADM1 enhances tumor growth and metastasis of SCLC cells probably by activating PI3K/Akt and PKA pathways through its binding proteins at 4.1BM. Citation Format: Toko Funaki, Takeshi Ito, Yoshinori Murakami. Overexpression of CADM1 enhances malignant features of small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4509.

The activated β-integrin (CgβV) enhances RGD-binding and phagocytic capabilities of hemocytes in Crassostrea gigas

Integrins are an important family of cell receptors that can bind foreign particles and promote cell phagocytosis after they are activated. In the present study, a novel β integrin was identified from pacific oyster Crassostrea gigas with an extracellular domain, a single transmembrane segment, and a short cytoplasmic domain. It was phylogenetically clustered with phagocytosis-related insecta βV, and designated as CgβV. CgβV shared a conserved NPX[Y/F] motif related to integrin activation with other phagocytosis-related β integrins. The mRNA transcripts of CgβV were widely detected in oyster tissues including hemocytes, gonad, adductor muscle, mantle, gill, and hepatopancreas, and the expression level in hemocytes was significantly up-regulated at 12 h after lipopolysaccharide (LPS) stimulation (p < 0.05), which was 2.29-fold higher than that in the control group. CgβV proteins were mainly observed on the hemocytes surface. The oyster hemocytes were found to bind fluorescein isothiocyanate (FITC)-labeled Arg-Gly-Asp-containing peptides (RGDCPs), and the binding capability was significantly up-regulated with the peak percentage of 37.6% at 12 h post LPS stimulation, which was higher than that in the control group (8.8%, p < 0.05), suggesting the activation of RGD-binding integrins on oyster hemocytes surface. The label-free RGDCPs and anti-CgβV antibody inhibited the binding capability of hemocytes towards FITC-labeled RGDCPs, which were significant lower in RGD blocking group (7.4%, p < 0.05) and anti-CgβV blocking group (22.1%, p < 0.05) than that in the control group (37.6%), indicating that CgβV could be a RGD-binding integrin. Phagocytosis assay demonstrated that LPS could enhance the phagocytosis of hemocytes towards Escherichia coli and fluorescent beads with the phagocytic rate (PR) of 18.3% and 17.4%, and phagocytic index (PI) of 5.29 and 37.71, respectively, which were significant higher than that in the control group (11.0% and 3.65 for E. coli, 9.8% and 29.26 for fluorescent beads, respectively, p < 0.05). In addition, both the label-free RGDCPs and anti-CgβV antibody significantly hindered the phagocytosis of hemocytes towards E. coli and fluorescent beads. After the E. coli and fluorescent beads were opsonized by oyster serum, the label-free RGDCPs still inhibited the phagocytosis of hemocytes towards them, while the anti-CgβV antibody could only inhibit the phagocytosis of hemocytes towards E. coli, suggesting that only the activated CgβV was involved in the enhancing phagocytosis for bacteria in oyster. Moreover, the key components of conserved integrin-mediated phagocytosis pathway including GTPases, talin proteins, Ca2+ and cAMP were all induced by LPS in hemocytes of oyster. All these results suggested that the activated CgβV enhanced RGD-binding and phagocytic capabilities of hemocytes, shedding lights on the mechanisms of integrin-mediated phagocytosis in mollusks.

The other face of MUC1 protein in cancer progression – the role of MUC1-C subunit

MUC1 is high-molecular-weight glycoprotein which is widely expressed on epithelial cell layers of various organs and is aberrantly overexpressed in most human carcinomas such as breast, ovarian or colon cancer. MUC1 contains three domains: short cytoplasmic and transmembrane domains, and a long extracellular domain. The protein is translated as a single polypeptide that undergoes auto-cleavage into two subunits: MUC1-N and MUC1-C, that form a stable noncovalent complex at the cell membrane. In normal epithelial cells, MUC1 expression is restricted to the apical side of the cells. During cancer transformation there is a loss of cell polarity and MUC1-C is found over the entire cell surface while MUC1-N is shed from the surface of carcinoma cells. As the result of depolarization, MUC1-C interacts with receptor tyrosine kinases (RTKs), such as EGFR and activates intracellular pathways which are important for cancer progression. Overexpression of MUC1 is also associated with the accumulation of MUC1-C in the cytoplasm and targeting of this subunit to the nucleus or mitochondria. Nuclear localization of MUC1-C is associated with activation of gene families involved in oncogenesis or cancer metabolism. Other results have shown that MUC1-C localizes to the mitochondrial outer membrane, where it blocks stress induced apoptosis. Recent studies have been directed at the MUC1-C subunit which reminds to be attractive target for the development of anti-cancer agents. In this review, the role of MUC1-C in cancer progression is discussed.

Some Aspects in Mechano-Biology of Platelet and Leukocyte in Blood Flows

For hemostasis and thrombosis, some proteins, such as Von Willebrand Factor (VWF, a multimeric plasma glycoprotein synthesized in endothelial cells and megakaryocytes and secreted to circulation or attached to endothelial cells), the metalloprotease ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13), P-selectin (one of three selectin family members with a N-terminal C-type lectin domain, an epidermal growth factor (EGF)-like module, a series of consensus repeat (CR) units, a transmembrane segment and a short cyto-plasmic domain) and β2 integrin. In adhesion and aggregation of circulating platelets towards to the sites of vascular injury, VWF on vascular wall captures and activates the circulating platelets through interaction with platelet receptor glycoprotein Ibα (GPIbα). The activated platelets will secrete P-selectin, mediating flowing leukocytes to be captured first to and then rolled on the platelets. Activation of β2 integrin on leukocytes makes the rolling cells slow down and adhere firmly to platelet. Pathological hemodynamic environment may cause platelet-induced inflammation overreaction of leukocytes, leading to mechanical instability of thrombotic plaque. Above mentioned events all are referred to their respective un-well known mechano-chemistry processes. For better understanding on the mechano-chemistry mechanism of interaction of leukocyte with platelet under flows, we have investigated the force-dependent Structure-function of VWF-A domain, force-regulated cleavage of A2 domain of von Willebrand factor (vWF) by ADAMTS13, P-selectin secretion from activated platelet, and P-selectin-mediated Activation of β2 integrin on leukocytes under shear stresses, and so on through AFM and flow chamber experiments and molecular dynamics simulation. Our data showed that, these events mentioned above were biphasic force-dependent. Increasing force stabilizes the globular VWF-A conformation first and then makes it become a spread one with higher affinity with platelet receptor glycoprotein Ibα (GPIbα), the force-regulated cleavage of VWF-A2 domain by ADAMTS13 maybe closely related to the induced-fit of ADMATS13 and VWF-A2, Force triggers and quickens P-selectin-induced Activation of β2 integrin on leukocytes.

MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth.

Intrinsically disordered proteins (IDPs)/intrinsically unstructured proteins are characterized by the lack of fixed or stable tertiary structure, and are increasingly recognized as an important class of proteins with major roles in signal transduction and transcriptional regulation. In this study, we report the identification and functional characterization of a previously uncharacterized protein (UPF0258/KIAA1024), major intrinsically disordered Notch2-associated receptor 1 (MINAR1). While MINAR1 carries a single transmembrane domain and a short cytoplasmic domain, it has a large extracellular domain that shares no similarity with known protein sequences. Uncharacteristically, MINAR1 is a highly IDP with nearly 70% of its amino acids sequences unstructured. We demonstrate that MINAR1 physically interacts with Notch2 and its binding to Notch2 increases its stability and function. MINAR1 is widely expressed in various tissues including the epithelial cells of the breast and endothelial cells of blood vessels. MINAR1 plays a negative role in angiogenesis as it inhibits angiogenesis in cell culture and in mouse matrigel plug and zebrafish angiogenesis models. Furthermore, while MINAR1 is highly expressed in the normal human breast, its expression is significantly downregulated in advanced human breast cancer and its re-expression in breast cancer cells inhibited tumor growth. Our study demonstrates that MINAR1 is an IDP that negatively regulates angiogenesis and growth of breast cancer cells.

CEACAM1 is associated with recurrence after hepatectomy for colorectal liver metastasis

BackgroundCarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is re-expressed at the invasion front of colorectal cancer. CEACAM1 expression at metastatic sites remains to be investigated. The current study aims to clarify the association between CEACAM1 expression and recurrence after hepatectomy of colorectal liver metastasis and to address whether CEACAM1 induces tumor-initiating properties needed for growth at metastatic sites. MethodsImmunohistochemical analyses for CEACAM1 were performed in 67 patients with liver metastasis of colorectal cancer who had undergone curative hepatectomy. The risk factors for postoperative recurrence were calculated based on a CEACAM1 cytoplasmic domain isoform at the primary tumor invasion front. To investigate the effects of CEACAM1 cytoplasmic isoforms on HT29 and HCT116 colorectal cancer cells, Western blotting for CD44 and CD133, flow cytometry for ALDH1 activity, and soft-agar colony formation assay were performed. ResultsCEACAM1 long (CEACAM1-L) and short (CEACAM1-S) cytoplasmic domain isoforms are strongly expressed on cancer cells in the liver metastases. Enhanced CEACAM1-S expression in the state of CEACAM1-L dominance at the primary tumor invasion front was an independent factor for colorectal cancer recurrence after curative hepatectomy. CEACAM1-4S-transfected HT29 and HCT116 cells had significantly higher CD44 expression and ALDH1 activity and increased the growth in anchorage-independent condition. ConclusionsHigh expression of CEACAM1-S at the primary lesion invasion front is associated with recurrence and prognosis of patients with colorectal liver metastasis after curative hepatectomy. The expression of CEACAM1-4S enhances the tumor-initiating property of colorectal cancer cells.

OR12 Integrin β1 partners with HLA class II molecules to mediate endothelial cell (EC) functions

Aim Engagement of cell surface HLA class II (HLA-II) regulates multiple cell functions through activating signal transduction pathways. Class II molecules are heterodimers consisting of two transmembrane glycoproteins with short cytoplasmic domains of 12–15 amino acids that lack kinase activity. We hypothesized that ligation of class II molecules by antibodies (Ab) recruits integrin β1 (ITGB1) into a complex with HLA-II to regulate signal transduction and cell function. Methods Class II expression was induced by adenoviral transfection of EC with CIITA. Western blots were used to quantitate protein phosphorylation and protein expression. The interactions between ITGB1 and class II were determined by coimmunoprecipitation (co-IP). BrdU assay was used to measure cell proliferation. Wound healing assay and transmigration assays were applied to determine class II-induced cell migration. Results Class II ligation-activated phosphorylation of Src, Erk and Akt was inhibited by siRNA depletion of ITGB1, but not by ITGB3, B4 and B5 siRNA. Class II-induced EC proliferation and migration were also attenuated by ITGB1 siRNA. Co-IP of class II molecules demonstrated that HLA-II Ab increased ITGB1 expression in the complex. Reverse IP showed that HLA-II was recruited into ITGB1 complex. ITGB1 siRNA decreased complex formation between HLA-II and ITGB1. Inhibiting Src and ERK kinases with PP2 and U0126, respectively, decreased class II-induced ITGB1 phosphorylation at Y783 (Fig). Treatment of EC with PP2 or U0126 reduced complex formation between HLA-II and ITGB1 (Fig) and Talin. Conclusion These results show that HLA-II and ITGB1 cooperate to trigger intracellular signaling in EC and stimulate the proliferation and migration of endothelial cells, which may be important in promoting antibody-mediated rejection (AMR) and transplant vasculopathy. These results provide a mechanistic understanding of the role of ITGB1 and HLA class II in AMR and may lead to the development of novel therapeutic strategies to prevent AMR. Download : Download high-res image (401KB) Download : Download full-size image

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria.

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.

9: RAGE is a cell surface nucleic acid sensor

RAGE is a multi-ligand cell surface receptor of the Immunoglobulin super-family. It is composed of three extracellular immunoglobulin-like domains (V, C1 and C2), a single trans-membrane domain and a short cytoplasmic domain. RAGE is predominately expressed in the lung under normal conditions. However, during inflammation, RAGE expression increases dramatically in tissues like the vascular system, CNS and some hematopoietic cells. RAGE ligands are structurally heterogeneous and bind to RAGE through sequence nonspecific electrostatic interactions. They include advanced glycation end products, the DNA binding protein HMGB1, several proteins of the S100 family and certain types of β -sheet fibrils such as β -amyloid. Hence, RAGE is involved in many chronic inflammatory diseases such as diabetes, Alzheimer’s disease, asthma and systemic lupus erythematosus. Using EMSA and AlphaScreen, we showed that DNA and RNA bind to RAGE in a sequence nonspecific manner through electrostatic interaction of their phosphate backbone with the V and C1 domains. Human embryonic kidney (HEK) cells expressing RAGE bound more fluorescently labelled RNA and DNA than non-expressing cells as shown by microscopy and flow cytometry. In order to investigate the effect of RAGE expression on nucleic acid (NA) sensing by endosomal TLRs, we used HEK cells expressing TLR7, TLR8, TLR13 (ssRNA) or TLR9 (DNA) together with RAGE and found that RAGE increased NA induced activation of the transcription factor NF κ B. Finally, in a mouse model of acute lung inflammation, DNA inhalation induced a strong inflammatory response in WT mice, while RAGE knockout mice displayed dramatically reduced inflammation. We show that RAGE binds NAs at the cell surface, increases their uptake and thereby amplifies the activation of endosomal TLRs. This work helps to further elucidate the function of RAGE in the development and perpetuation of chronic inflammation and presents RAGE as a promising target in the development of anti-inflammatory treatments.