This study shows how chronic stress and heat shock response exacerbate the phenotype in protein misfolding diseases by triggering a Maladaptive Stress Response; this pathway represents a promising therapeutic target for multiple genetic disorders.

Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, composed of the plasma membrane t-SNAREs syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of 2 membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration. Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and both continuous wave (CW) and pulsed EPR revealed that cholesterol induces a conformational change of the v-SNARE transmembrane domain (TMD) from an open scissors-like dimer to a parallel dimer. When the TMD was forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissors-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor.EPR ͉ membrane fusion ͉ syntaxin ͉ synaptobrevin I n the neuron, synaptic vesicles fuse with plasma membrane to release neurotransmitters into the synaptic cleft (1-4). Membrane fusion involves extensive bilayer remodeling, which requires free energy or an effective catalyst (5-6). SNAREs (soluble Nethylmaleimide-sensitive factor attachment protein receptors) are believed to be the fusion machine that serves as the energy source (7-8) or catalyst that lowers the fusion energy barrier (9). At the onset of fusion, vesicle associated (v-) SNARE synaptobrevin (or VAMP2) combines with target membrane (t-) SNAREs Syntaxin 1A and SNAP-25 to form a helical bundle that bridges 2 membranes, facilitating fusion (10-14).Cholesterol is a major component of the plasma and the vesicle membranes, composing as much as 30-40 mol % of the total lipids (15-16). Cholesterol is structurally rigid and known to harden the membrane significantly, making it resistant to being bent or deformed. However, there is evidence that cholesterol accelerates membrane fusion that involves a series of bilayer deformations (17)(18)(19)(20). The physical origin of cholesterol's positive role can be found partly in its spontaneous negative curvature such that it prefers to be in the negatively curved (concave) surface but dislikes being in the positively curved (convex) surface (21).Despite progress in understanding the effects of cholesterol on the bilayer curvature, not much is known about its influence on the lateral distribution of SNAREs in the membrane or on structures of their transmembrane domains (TMDs). A series of recent reports addresses the importance of the clustering of SNAREs at the fusion site for successful fusion (19,(22)(23)(24). In addition, there is evidence that the SNARE TMDs are essential for formation of the fusion pore (25-27). Considering cholesterol...

The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin N-terminal homology (ENTH) domain has potent vesicle tubulation activity despite a lack of intrinsic molecular curvature. EPR revealed that the N-terminal ␣-helix penetrates the phosphatidylinositol 4,5-bisphosphate-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin 1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.Cell membranes undergo dynamic structural changes and remodeling during movement, division, and vesicle trafficking (1, 2). In particular, vesicle budding and fusion constantly take place in various cell membranes to maintain communication and transport between membrane-bound compartments (3). Dynamic membrane remodeling involves changes in local membrane curvature (or deformation) that are orchestrated by membrane lipids, integral membrane proteins, and cytoskeletal proteins (4, 5). Recently, several groups of cytosolic proteins that reversibly bind membranes and induce and/or detect different types of membrane curvatures during membrane remodeling have been identified. In particular, cytosolic proteins that are involved in different stages of clathrin-mediated endocytosis have received the most attention (6, 7), and many of them contain either an Ap180 N-terminal homology (ANTH) 2 /epsin N-terminal homology (ENTH) (8, 9) or a Bin-amphiphysin-Rvs (BAR) domain (9 -12). Although ENTH (13) and BAR domains (14 -17) have been reported to have in vitro vesicle-tubulating activities, the exact mechanisms by which these domains induce membrane deformation and larger scale membrane remodeling, especially under physiological conditions, are yet to be elucidated. For BAR domains, their unique intrinsic molecular curvatures have been postulated to be important for membrane deformation through a scaffolding mechanism (4). Also, recent studies have shown that F-BAR domains from FBP17 and CIP4 form highly ordered self-assembly in two-dimensional (18) and three-dimensional (19) crystals and that disruption of intermolecular interactions abrogates their membrane deformation activities. Despite remarkable success in structural characterization of various BAR and ENTH domains, questions still remain as to whether individual domains function by a universal mechanism or by different mechanisms, whether the intact proteins harborin...

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Lung cancer remains to be the leading cause of cancer-related mortality worldwide. Finding new noninvasive biomarkers for lung cancer is still a significant clinical challenge. Exosomes are membrane-bound, nano-sized vesicles that are released by various living cells. Studies on exosomal proteomics may provide clues for developing clinical assays. In this study, we performed semi-quantitative proteomic analysis of proteins that were purified from exosomes of NCI-H838 non-small cell lung cancer cell line, with total cellular membrane proteins as control. In the exosomes, LC-MS/MS by data-independent analysis mode identified 3235 proteins. THBS1, ANXA6, HIST1H4A, COL18A1, MDK, SRGN, ENO1, TUBA4A, SLC3A2, GPI, MIF, MUC1, TALDO1, SLC7A5, ICAM1, HSP90AA1, G6PD, and LRP1 were found to be expressed in exosomes at more than 5-fold higher level as compared to total cellular membrane proteins. A well-known cancer biomarker, MUC1, is expressed at 8.98-fold higher in exosomes than total cellular membrane proteins. Subsequent analysis of plasma exosomes from non-small cell lung cancer (NSCLC) patients by a commercial electrochemiluminescence immunoassay showed that exosomal MUC1 level is 1.5-fold higher than healthy individuals (mean value 1.55 ± 0.16 versus mean value 1.05 ± 0.06, p = 0.0213). In contrast, no significant difference of MUC1 level was found between NSCLC patients and healthy individuals′ plasma (mean value 5.48 ± 0.65 versus mean value 4.16 ± 0.49). These results suggest that certain proteins, such as MUC1, are selectively enriched in the exosome compartment. The mechanisms for their preferential localization and their biological roles remain to be studied.

Disease and Gene Annotations database (DGA, http://dga.nubic.northwestern.edu) is a collaborative effort aiming to provide a comprehensive and integrative annotation of the human genes in disease network context by integrating computable controlled vocabulary of the Disease Ontology (DO version 3 revision 2510, which has 8043 inherited, developmental and acquired human diseases), NCBI Gene Reference Into Function (GeneRIF) and molecular interaction network (MIN). DGA integrates these resources together using semantic mappings to build an integrative set of disease-to-gene and gene-to-gene relationships with excellent coverage based on current knowledge. DGA is kept current by periodically reparsing DO, GeneRIF, and MINs. DGA provides a user-friendly and interactive web interface system enabling users to efficiently query, download and visualize the DO tree structure and annotations as a tree, a network graph or a tabular list. To facilitate integrative analysis, DGA provides a web service Application Programming Interface for integration with external analytic tools.

Hepatitis B virus (HBV)-encoded X protein (HBx) plays a critical role in HBV-related hepatocarcinoma development. In this study, we demonstrate that HBx is specifically modified by NEDD8. We found that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability by preventing its ubiquitinationmediated degradation. Consistently, analysis of 160 hepatocellular carcinoma patient specimens indicated that the amount of HDM2 protein correlates with HBx protein level. We identified that HBx K91 and K95 as the key HBx NEDDylation sites and observed that the NEDDylation-deficient HBx has shorter half-life. We generated Huh7 cell lines which ectopically express wild-type and NEDDylation-deficient HBx and found that NEDDylation-deficient HBx showed less chromatin localization and less DDB1 binding. Consistently, the expression of HBx-regulated genes (IL-8, MMP9, and YAP) and HBV transcription (the activity of HBV enhancer and the amount of pgRNA transcribed from cccDNA) were significantly higher in cells expressing wild-type (WT) HBx than that in cells expressing mutant HBx. In addition, HBx-expressing cells proliferated faster than control and mutant HBx-expressing cells. We also showed that the ability of WT HBx-expressing cells to form tumors in nude mice was significantly higher than that of mutant HBx-expressing cells. In conclusion, we revealed that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability and chromatin localization, which in turn favors HBx-dependent transcriptional regulation, cell proliferation, and HBV-driven tumor growth. IMPORTANCE Hepatitis B virus (HBV) HBx protein plays a critical role in viral replication and hepatocarcinogenesis. However, the regulation of HBx stability is not well understood. We found that HBx is modified by NEDD8 and that the HDM2 E3 ligase promotes HBx NEDDylation to enhance HBx stability by inhibiting its ubiquitination. We provide a new evidence to show the positive correlation between HDM2 and HBx in clinical hepatocellular carcinoma (HCC) samples. We also identified the major NEDDylation sites on HBx. Our studies indicate that the defective NEDDylation of HBx negatively affects its ability to activate the transcription of downstream genes and promote cell proliferation and tumor growth in vivo. Taken together, our findings reveal a novel posttranslational modification of HBx by HDM2 which regulates its stability, subcellular localization, and functions. These findings indicate that HDM2 is an important regulator on HBx and a potential diagnosis/therapeutic marker for HBVassociated HCC.KEYWORDS HBx, NEDDylation, stability, chromatin localization, hepatocellular carcinoma, HDM2, hepatitis B virus

Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. In this study, we found that HBV inhibited the chemotherapy drug etoposide-induced apoptosis of hepatoma cells. Further analysis revealed that HBV mRNAs possess a microRNA 15a/16 (miR-15a/16)-complementary site (HBV nucleotides [nt] 1362 to 1383) that acts as a sponge to bind and sequester endogenous miR-15a/16. Consequently, Bcl-2, known as the target of miR-15a/16, was upregulated in HBV-infected cells. The data from HBV-transgenic mice further confirmed that HBV transcripts cause the reduction of miR15a/16 and increase of Bcl-2. More importantly, we examined the levels of HBV transcripts and miR-15a/16 in HBV-infected HCC from patients and found that the amount of HBV mRNA and the level of miR-15a/16 were negatively correlated. Consistently, the level of Bcl-2 mRNA was upregulated in HBV-infected patients. In conclusion, we identified a novel HBV mRNAmiR-15a/16 -Bcl-2 regulatory pathway that is involved in inhibiting etoposide-induced apoptosis of hepatoma cells, which may contribute to facilitating chronic HBV infection and hepatoma development. There are approximately 350 million chronic hepatitis B virus (HBV) carriers worldwide, and chronic HBV infection is the major etiological factor in hepatocellular carcinoma (HCC) (1, 2). The relative risk for the development of HCC in chronic hepatitis B (CHB) patients is estimated to be 25 to 37 times higher than that in those without infection (1,3,4).HBV is an enveloped, partially double-stranded DNA virus with a genome size of 3.2 kb. The HBV genome contains four overlapping open reading frames (ORFs). The RNA transcripts are polyadenylated and capped and are named the pre-C/C or pregenomic RNA (pgRNA) and the pre-S, S, and X mRNAs. These mRNAs encode several viral proteins, including the polymerase, core, HBe, pre-S1, S2, S, and X proteins (5). HBV has been reported to play an important role in regulating apoptosis. For example, HBV core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression (6). HBx can bind to the C terminus of p53 and inhibit p53-mediated cellular processes, including transcriptional transactivation and apoptosis (7). But the HBx protein was also found to sensitize cells to apoptotic killing by tumor necrosis factor alpha (8) and to inhibit Fas-mediated apoptosis associated with upregulation of the SAPK/JNK pathway in Chang cells (9).MicroRNAs (miRNAs) are single-stranded noncoding RNAs which negatively regulate gene expression at the posttranscriptional level, primarily through base pairing to the 3=-untranslated region (UTR) of target mRNA (10). Growing evidence indicates that microRNAs control basic cell functions, ranging from proliferation to apoptosis, by direct targeting (11,12). For instance, miR-101 exerts a proapoptotic function by targeting Mcl-1 (13), and miR-29c inhibits ce...