Hypertonia-linked protein Trak1 functions with mitofusins to promote mitochondrial tethering and fusion

Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson's disease, dystonia, and epilepsy. Genetic studies have identified a homozygous truncation mutation in Trak1 that causes hypertonia in mice. Moreover, elevated Trak1 protein expression is associated with several types of cancers and variants in Trak1 are linked to childhood absence epilepsy in humans. Despite the importance of Trak1 in health and disease, the mechanisms of Trak1 action remain unclear and the pathogenic effects of Trak1 mutation are unknown. Here we report that Trak1 has a crucial function in regulation of mitochondrial fusion. Depletion of Trak1 inhibits mitochondrial fusion, resulting in mitochondrial fragmentation, whereas overexpression of Trak1 elongates and enlarges mitochondria. Our analyses revealed that Trak1 interacts and colocalizes with mitofusins on the outer mitochondrial membrane and functions with mitofusins to promote mitochondrial tethering and fusion. Furthermore, Trak1 is required for stress-induced mitochondrial hyperfusion and pro-survival response. We found that hypertonia-associated mutation impairs Trak1 mitochondrial localization and its ability to facilitate mitochondrial tethering and fusion. Our findings uncover a novel function of Trak1 as a regulator of mitochondrial fusion and provide evidence linking dysregulated mitochondrial dynamics to hypertonia pathogenesis.

Significance of Vesicle-Associated Membrane Protein 8 Expression in Predicting Survival in Breast Cancer / 한국유방암학회지

PURPOSE: Vesicle-associated membrane protein 8 (VAMP8) is a soluble N-ethylmaleimide-sensitive factor receptor protein that participates in autophagy by directly regulating autophagosome membrane fusion and has been reported to be involved in tumor progression. Nevertheless, the expression and prognostic value of VAMP8 in breast cancer (BC) remain unknown. This study aimed to evaluate the clinical significance and biological function of VAMP8 in BC. METHODS: A total of 112 BC samples and 30 normal mammary gland samples were collected. The expression of VAMP8 was assessed in both BC tissues and normal mammary gland tissues via a two-step immunohistochemical detection method. RESULTS: The expression of VAMP8 in BC tissues was significantly higher than that in normal breast tissues. Furthermore, increased VAMP8 expression was significantly correlated with tumor size (p=0.007), lymph node metastasis (p=0.024) and recurrence (p=0.001). Patients with high VAMP8 expression had significantly lower cumulative recurrence-free survival and overall survival (p < 0.001 for both) than patients with low VAMP8 expression. In multivariate logistic regression and Cox regression analyses, lymph node metastasis and VAMP8 expression were independent prognostic factors for BC. CONCLUSION: VAMP8 is significantly upregulated in human BC tissues and can thus be a practical and potentially effective surrogate marker for survival in BC patients.

Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagy-inducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM's inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.

Strategy for Developing Medical Arsenals by Modulation of Membrane Fusion Activity of Influenza Virus Hemagglutinin

Influenza virus is a serious pathogen that burdens society with health care costs, and can lead to fatality. The virus is dealt with currently by vaccination and anti-influenza drugs. However, vaccines need to be improved towards safer and more efficient production formats, and drugs need to be constantly renewed to cope with resistances. That the neuraminidase inhibitors are only drugs currently available warrants urgent attention to an alternative anti-influenza target. In this paper we introduce studies on fusion activity of influenza virus hemagglutinin (HA), and discuss how to best utilize the knowledge for an improved vaccine development and an anti-influenza drug search. Potential application of mutations resulting in changes in fusion activity to cell culture optimized vaccine virus development and strategies to develop broad spectrum anti-influenza drugs through targeting the conserved fusion domain of the HA are discussed.

Research progress of the molecule mechanisms of Ebola virus infection of cells / 病毒学报

Ebola virus can cause severe Ebola hemorrhagic fever. The mortality rate is 90 percent. Up till now, there is no effective vaccine or treatment of Ebola virus infection. Relaed researches on Ebola virus have become a hot topic in virology. The understanding of molecular mechanisms of Ebola virus infection of cells are important for the development of vaccine and anti-virus drugs. Therefore, this review summarized the recent research progress on the mechanisms of Ebola virus infection.

The receptors and entry of measles virus: a review / 生物工程学报

Measles virus is an enveloped virus with a non-segmented negative-sense RNA genome. Two envelope glycoproteins on the viral surface, namely hemagglutinin (H) and membrane fusion protein (F), are responsible for the virus entry into susceptible host cells. The specific interaction between H and its cellular receptors is a key step in successful virus infection, determining the infectivity and tissue tropism of the measles virus. Thus far, three H receptors have been identified, including the complement regulatory molecule CD46, the signaling lymphocyte activation molecule (SLAM) and the cell adhesion molecule Nectin-4. Here, we reviewed our molecular understanding on the recognition mechanism of these receptors by the viral H protein, aiming to promote future studies on antiviral drug design and measles virus-based oncolytic therapy.

An Universal Approach to Getting Ahead for Influenza B Vaccines

Cross-reactive neutralizing antibodies against influenza A viruses have received attention for their potentials for prophylactic and therapeutic. These antibodies usually bind to relatively conserved stem domains of influenza hemagglutinin, one of surface glycoproteins responsible for viral binding to sialic acid-tagged cellular receptors and for membrane fusion to initiate a release process of viral genomes inside cells. Recently, a similar approach extended to influenza B viruses, which causing annual epidemics only in the human population, and some of human monoclonal antibodies exhibited promising efficacies against two antigenically diverged lineages of influenza B viruses. Moreover, one of these broadly neutralizing antibodies protected mice against both of influenza A and B challenges. Appropriate immunization may selectively enhance the efficacy of these antibodies, and this strategy may lead individuals to be prepared with broad immune responses against various influenza viruses.

Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R(696) (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R(696), it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R(696) with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.

Prokaryotic expression of S2 extracellular domain of SARS coronavirus spike protein and its fusion with Hela cell membrane / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.</p><p><b>METHODS</b>S2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.</p><p><b>RESULTS</b>The pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.</p><p><b>CONCLUSIONS</b>The expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.</p>

Effects of disulfide bridges in glycoprotein E1 on the membrane fusion activity of rubella virus / 病毒学报

To reveal the effects of disulfide bridges in rubella virus glycoprotein E1 on the membrane fusion activity, the recombinant plasmid pBSK-SPE2E1 and site-directed mutagenesis to mutate 11 cysteines individually in the ectodomain of E1 to remove a disulfide bridge from the wild-type E1 were constructed. All mutants and the wild-type plasmid were expressed on BHK-21 cell. Giemsa Staining was used to show the polykaryon formed in the transfected BHK-21 cells. The cell surface expression efficiency of the plasmids was assayed with fluorescence-activated cell sorter (FACS). Hemadsorption was performed to detect the receptor recognition activity of the recombinant plasmids. The results showed that all the 10 disulfide bridges in the ectodomain of E1 played an important role in the process of the membrane fusion. The removal of any disulfide bridge resulted in the loss of the fusion activity. The disulfide formed by the 5th and the 8th cysteine might be critical for the interaction of E1 and E2. While the disulfide bridges formed by the 3rd, the 4th, and the 13th might influence the membrane fusion activity of E1 directly.

1,2,6-tri-O-galloyl-beta-D-glucopyranose inhibits gp41-mediated HIV envelope fusion with target cell membrane / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the inhibitory effect of 1,2,6-Tri-O-galloyl-beta-D-glucopyranose (TGGP) from Balanophora japonica Makino on human immunodeficiency virus (HIV) entry into the host cells and explore the mechanisms.</p><p><b>METHODS</b>TGGP was purified from Balanophora japonica Makino by n-hexane and ethyl acetate extraction and column chromatography. The inhibitory activity of TGGP on HIV gp41 six-helix bundle formation was measured with ELISA, N-PAGE and SE-HPLC, and the inhibitory effect of TGGP on HIV envelope grlycoprotein-induced cell-cell fusion was detected using a non-infectious cell-based assay.</p><p><b>RESULTS</b>TGGP inhibited HIV gp41 six-helix bundle formation, with an IC50 of 1.37-/+0.19 microg/ml as determined by ELISA, and this activity was further confirmed by N-PAGE and SE-HPLC. TGGP at 25 microg/ml significantly inhibited syncytium formation between the effector (CHO-WT) and the target (MT-2) cells.</p><p><b>CONCLUSION</b>The HIV transmembrane subunit gp41 mediates the entry of HIV into the target cells. TGGP can inhibit HIV fusion and entry into the target cells by inhibiting the formation of gp41 six-helix bundles, suggesting the potential of TGGP as a microbicide to prevent sexual transmission of HIV.</p>

Property of liposomal fusion induced by acid-sensitive polymer / 药学学报

The fusion between liposome-liposome, liposome-biomembarnes induced by acid-sensitive polymers has been systematically investigated. The polymer-liposomes were constructed by post-insertion method with the poly (2-ethylacrylic acid) (PEAA) alkylamide derivatives. The liposomal fusion was studied by use of fluorescence resonance energy transfer assay, particle size, fluorescent-photometer. The results indicated that the poly (2-ethylacrylic acid)-liposomes has very strong acidic induced fusion capability. Under acidic conditions, acid-sensitive polymer liposomes fused each other, the fusion closely related to the molecular weight of acid sensitivity polymer on the surface of liposomes. The acidic fusion of polymer-liposomes was dependent upon the lipids composition, the degree of fusion was reversely related to the cholesterol contents. Acid-en ci-nsitive polymer liposomes fused with erythrocyte ghosts. The liposomal fusion induced by acid-sensitive polymer associated with the increase of membrane permeability. The good acid-sensitivity of PEAA has been further demonstrated by membrane fusion in current experiments, and the liposomes prepared with lipid anchored-poly (2-ethylacrylic acid) were developeds s a potential pH sensitive delivery system.

Molecular correlates of syncytialization in muscle and placenta.

Syncytialization is one of the most fundamental processes in life. It is observed during development of muscle and osteoclast, and syncytiotrophoblast formation in placental villi. Syncytialization involves recognition, migration, adhesion and finally cell fusion between two interacting cells. It is an energy-dependent process which is essentially restricted to a small portion of interacting cellular membranes. Such regions of membranes may differ from other regions of cell surface in terms of physico-chemistry and expression of specific protein biomolecules resulting in restriction of this process to cells of specific competence. Despite the fact that membrane biologists have given significant quanta of efforts to understand the basic principle underlying this fundamental process of life, further large scale initiatives have to be undertaken to dissect the underlying molecular correlates central to this event.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Expression and purification of heptad repeat region of the mumps virus F protein and analysis of characteristics / 生物工程学报

Two Heptad repeat motifs (HR1 and HR2) from paramyxoviruses F protein could form thermostable heterodimers containing high alpha-helix while virus infected host cell. Following that the viral membrane and the host cell membrane were juxtaposed, which leads to membrane fusion. Mumps virus (MuV) is a member of the genus Rubulavirus in the family of Paramyxoviridae. MuV could use similar infection mechanism as well as other paramyxoviruses. In this study the HR1 and HR2 regions of MuV F protein were predicted by a computer program and expressed in E. coli with the GST fusion expression system. The GST fusion or GST-removed proteins were purified with Gluthathion Sepharose 4B Column. GST pull-down experiment suggested the interaction of HR1 and HR2 peptides, and analysis of gel filtration showed two peptides could form multimer, which indicates that the HR regions of MuV F protein may play an important role in virus fusion.

Calcium - how and why?

Calcium is among the most commonly used ions, in a multitude of biological functions, so much so that it is impossible to imagine life without calcium. In this article I have attempted to address the question as to how calcium has achieved this status with a brief mention of the history of calcium research in biology. It appears that during the origin and early evolution of life the Ca2+ ion was given a unique opportunity to be used in several biological processes because of its unusual physical and chemical properties.

Electron microscopic study of adhesion between Helicobacter pylori and gastric epithelial cell / 대한내과학회지

BACKGROUND: The purpose of this study was to investigate the ultrastructural relation of H. pylori and gastric epithelial cells in their adhesion. METHODS: Endoscopic biopsy of gastric antrum and body was performed from 15 patients (9 men, 6 women) with chronic gastritis. These specimens were processed and observed by transmission electron microscope (Hitachi H-600). RESULTS: On the basis of morphological appearances, the different types of adhesion of the organism with the epithelial cells were categorized as filamentous connection, adhesion pedestals, membrane fusion. Coccoid and intermediate forms were associated with filamentous connection whereas bacillary forms were associated with adhesion pedestals and membrane fusion. CONCLUSION: Various types of adhesion were associated with H. pylori and gastric epithelium. Further studies are needed to investigate biophysiologic influence to epithelial cells by ultrastructural relationship.(Korean J Med 60:16-21, 2001)

Identification of novel membrane structures in Plasmodium falciparum infected erythrocytes

Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocytes membrane, the parasitophorous vacuolar membrane and the residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastrutural studies show that a novel structure extends from the former parasite compartment to the membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell.