SEC22B inhibition attenuates colorectal cancer aggressiveness and autophagic flux under unfavorable environment.

Autophagy has bidirectional functions in cancer by facilitating cell survival and death in a context-dependent manner. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a large family of proteins essential for numerous biological processes, including autophagy; nevertheless, their potential function in cancer malignancy remains unclear. Here, we explored the gene expression patterns of SNAREs in tissues of patients with colorectal cancer (CRC) and discovered that SEC22B expression, a vesicle SNARE, was higher in tumor tissues than in normal tissues, with a more significant increase in metastatic tissues. Interestingly, SEC22B knockdown dramatically decreased CRC cell survival and growth, especially under stressful conditions, such as hypoxia and serum starvation, and decreased the number of stress-induced autophagic vacuoles. Moreover, SEC22B knockdown successfully attenuated liver metastasis in a CRC cell xenograft mouse model, with histological signs of decreased autophagic flux and proliferation within cancer cells. Together, this study posits that SEC22B plays a crucial role in enhancing the aggressiveness of CRC cells, suggesting that SEC22B might be an attractive therapeutic target for CRC.

A Genome-Wide Screen Reveals That Endocytic Genes Are Important for Pma1p Asymmetry during Cell Division in Saccharomyces cerevisiae.

An asymmetry in cytosolic pH between mother and daughter cells was reported to underlie cellular aging in the budding yeast Saccharomyces cerevisiae; however, the underlying mechanism remains unknown. Preferential accumulation of Pma1p, which pumps cytoplasmic protons out of cells, at the plasma membrane of mother cells, but not of their newly-formed daughter cells, is believed to be responsible for the pH increase in mother cells by reducing the level of cytoplasmic protons. This, in turn, decreases the acidity of vacuoles, which is well correlated with aging of yeast cells. In this study, to identify genes that regulate the preferential accumulation of Pma1p in mother cells, we performed a genome-wide screen using a collection of single gene deletion yeast strains. A subset of genes involved in the endocytic pathway, such as VPS8, VPS9, and VPS21, was important for Pma1p accumulation. Unexpectedly, however, there was little correlation between deletion of each of these genes and the replicative lifespan of yeast, suggesting that Pma1p accumulation in mother cells is not the key determinant that underlies aging of mother cells.

Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments.

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

SPIN90, an adaptor protein, alters the proximity between Rab5 and Gapex5 and facilitates Rab5 activation during EGF endocytosis.

During ligand-mediated receptor endocytosis, the small GTPase Rab5 functions in vesicle fusion and trafficking. Rab5 activation is known to require interactions with its guanine nucleotide-exchange factors (GEFs); however, the mechanism regulating Rab5 interactions with GEFs remains unclear. Here, we show that the SH3-adapter protein SPIN90 participates in the activation of Rab5 through the recruitment of both Rab5 and its GEF, Gapex5, to endosomal membranes during epidermal growth factor (EGF)-mediated endocytosis. SPIN90 strongly interacts with the inactive Rab5/GDI2 complex through its C-terminus. In response to EGF signaling, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of SPIN90 at Thr-242 enables SPIN90 to bind Gapex5 through its N-terminal SH3 domain. Gapex5 is a determinant of Rab5 membrane targeting, while SPIN90 mediates the interaction between Gapex5 and Rab5 in a phosphorylation-dependent manner. Collectively, our findings suggest that SPIN90, as an adaptor protein, simultaneously binds inactive Rab5 and Gapex5, thereby altering their spatial proximity and facilitating Rab5 activation.

TAGLN2 polymerizes G-actin in a low ionic state but blocks Arp2/3-nucleated actin branching in physiological conditions.

TAGLN is an actin-binding protein family that comprises three isoforms with theorized roles in smooth muscle differentiation, tumour development, lymphocyte activation, and brain chemistry. However, their fundamental characteristics in regulation of the actin-based cytoskeleton are not fully understood. Here we show that TAGLN2 (including TAGLN1 and TAGLN3) extensively nucleates G-actin polymerization under low-salt conditions, where polymerization would be completely suppressed. The calponin homology domain and actin-binding loop are essential to mechanically connect two adjacent G-actins, thereby mediating multimeric interactions. However, TAGLN2 blocked the Arp2/3 complex binding to actin filaments under physiological salt conditions, thereby inhibiting branched actin nucleation. In HeLa and T cells, TAGLN2 enhanced filopodium-like membrane protrusion. Collectively, the dual functional nature of TAGLN2-G-actin polymerization and Arp2/3 complex inhibition-may account for the mechanisms of filopodia development at the edge of Arp2/3-rich lamellipodia in various cell types.

Strategies to Tackle Radiation Resistance by Penetrating Cancer Stem Cell Line of Scrimmage.

BACKGROUND: Radiation therapy has become a common therapy for cancer patients and largely improved therapeutic outcomes. However, recurrence after radiation therapy is still a major unsolved concern of cancer patients. The Cancer Stem Cells (CSCs), a small sub-population with the capacity of self-renewal and differentiation into diverse types of cancer cells, are considered as strong drivers of metastasis, resistance, and recurrence. OBJECTIVE: We overview the mechanisms behind the radiation resistance of CSCs, and introduce recent patents with the potential to overcome such an obstacle. In this review article, we will introduce those patented compounds as potential combinatory drugs for radiation therapy and present their specific molecular mechanisms. METHOD: From a thorough literature review on the mechanisms of radiation resistance exhibited by CSCs, several patents that have disclosed novel compounds or their specific applications for improving radiation therapy will be introduced with their developmental status and experimental results. RESULTS: Recent studies have focused on revealing the molecular mechanisms of CSC contributing to radiation resistance and succeeded to discover that CSCs possess intrinsic and extrinsic resistance by preventing radiation-induced cellular damages and by adapting to the changes occurred by radiation exposure, respectively. Among currently discovered resistant mechanisms of CSCs, DNA repair and redox system directly decrease radiation-induced DNA breaks and relieve cellular stress caused by Reactive Oxygen Species (ROS). Indirectly, altered metabolism and cellular signaling may help CSCs adapt to the desolate microenvironment disrupted by radiation exposure. In order to overcome this obstacle on the road to the efficient cancer treatment, diverse therapeutic strategies using anti-sense oligonucleotides, small molecule inhibitors and antibodies have been tested and some of them actually showed an efficacy on improving current radiation therapy. CONCLUSION: Many agents that directly or indirectly sensitize CSCs have been developed and some of them are on clinical trial. We expect that they will be used to enhance clinical radiation therapy in the future.

Roles of Wnt Target Genes in the Journey of Cancer Stem Cells.

The importance of Wnt/ß-catenin signaling in cancer stem cells (CSCs) has been acknowledged; however, the mechanism through which it regulates the biological function of CSCs and promotes cancer progression remains elusive. Hence, to understand the intricate mechanism by which Wnt controls stemness, the specific downstream target genes of Wnt were established by analyzing the genetic signatures of multiple types of metastatic cancers based on gene set enrichment. By focusing on the molecular function of Wnt target genes, the biological roles of Wnt were interpreted in terms of CSC dynamics from initiation to metastasis. Wnt signaling participates in cancer initiation by generating CSCs from normal stem cells or non-CSCs and augmenting persistent growth at the primary region, which is resistant to anti-cancer therapy. Moreover, it assists CSCs in invading nearby tissues and in entering the blood stream, during which the negative feedback of the Wnt signaling pathway maintains CSCs in a dormant state that is suitable for survival. When CSCs arrive at distant organs, another burst of Wnt signaling induces CSCs to succeed in re-initiation and colonization. This comprehensive understanding of Wnt target genes provides a plausible explanation for how Wnt allows CSCs variation during cancer progression.

Structural insights into the interaction of human p97 N-terminal domain and SHP motif in Derlin-1 rhomboid pseudoprotease.

The interaction of the rhomboid pseudoprotease Derlin-1 and p97 is crucial for the retrotranslocation of polyubiquitinated substrates in the endoplasmic reticulum-associated degradation pathway. We report a 2.25 Å resolution structure of the p97 N-terminal domain (p97N) in complex with the Derlin-1 SHP motif. Remarkably, the SHP motif adopts a short, antiparallel ß-strand that interacts with the ß-sheet of p97N-a site distinct from that to which most p97 adaptor proteins bind. Mutational and biochemical analyses contributed to defining the specific interaction, demonstrating the importance of a highly conserved binding pocket on p97N and a signature motif on SHP. Our findings may also provide insights into the interactions between other SHP-containing proteins and p97N.

Bioengineered yeast-derived vacuoles with enhanced tissue-penetrating ability for targeted cancer therapy.

Despite the appreciable success of synthetic nanomaterials for targeted cancer therapy in preclinical studies, technical challenges involving their large-scale, cost-effective production and intrinsic toxicity associated with the materials, as well as their inability to penetrate tumor tissues deeply, limit their clinical translation. Here, we describe biologically derived nanocarriers developed from a bioengineered yeast strain that may overcome such impediments. The budding yeast Saccharomyces cerevisiae was genetically engineered to produce nanosized vacuoles displaying human epidermal growth factor receptor 2 (HER2)-specific affibody for active targeting. These nanosized vacuoles efficiently loaded the anticancer drug doxorubicin (Dox) and were effectively endocytosed by cultured cancer cells. Their cancer-targeting ability, along with their unique endomembrane compositions, significantly enhanced drug penetration in multicellular cultures and improved drug distribution in a tumor xenograft. Furthermore, Dox-loaded vacuoles successfully prevented tumor growth without eliciting any prolonged immune responses. The current study provides a platform technology for generating cancer-specific, tissue-penetrating, safe, and scalable biological nanoparticles for targeted cancer therapy.

SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae.

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene.

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.

Peroxisome-localized hepatitis Bx protein increases the invasion property of hepatocellular carcinoma cells.

HBx acts as a multifunctional regulator that modulates various cellular responses, which can lead to development and progression of hepatocellular carcinoma (HCC). Here, we show that the HBx protein is also localized to peroxisomes, and this increases cellular reactive oxygen species (ROS) to levels that are higher than when HBx is localized to other organelles. The elevated ROS strongly activated nuclear factor (NF)-κB. In addition, the peroxisome-localized HBx increased the expressions of matrix metalloproteinases and decreased the expression of E-cadherin, which increased the invasive ability of HCC cells. Thus, a specific distribution of HBx to peroxisomes may contribute to HCC progression by increasing the invasive ability of HCC cells through elevation of the cellular ROS level.

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion.

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms.

SPIN90 knockdown attenuates the formation and movement of endosomal vesicles in the early stages of epidermal growth factor receptor endocytosis.

The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation.

hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene.

Fas is a transmembrane cell surface protein recognized by Fas ligand (FasL). When FasL binds to Fas, the target cells undergo apoptosis. A soluble Fas molecule that lacks the transmembrane domain is produced from skipping of exon 6 encoding this region in alternative splicing procedure. The soluble Fas molecule has the opposite function of intact Fas molecule, protecting cells from apoptosis. Here we show that knockdown of hnRNP A1 promotes exon 6 skipping of Fas pre-mRNA, whereas overexpression of hnRNP A1 reduces exon 6 skipping. Based on the bioinformatics approach, we have hypothesized that hnRNP A1 functions through interrupting 5' splice site selection of exon 5 by interacting with its potential binding site close to 5' splice site of exon 5. Consistent with our hypothesis, we demonstrate that mutations of the hnRNP A1 binding site on exon 5 disrupted the effects of hnRNP A1 on exon 6 inclusion. RNA pull-down assay and then western blot analysis with hnRNP A1 antibody prove that hnRNP A1 contacts the potential binding site RNA sequence on exon 5 but not the mutant sequence. In addition, we show that the mutation of 5' splice site on exon 5 to a less conserved sequence destructed the effects of hnRNP A1 on exon 6 inclusion. Therefore we conclude that hnRNP A1 interacts with exon 5 to promote distal exon 6 inclusion of Fas pre-mRNA. Our study reveals a novel alternative splicing mechanism of Fas pre-mRNA.

Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation.

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.

Receptor-mediated ER export of human MHC class I molecules is regulated by the C-terminal single amino acid.

Major histocompatibility complex class I (MHC-I) molecules bind antigens in the endoplasmic reticulum (ER) and deliver them to the cell surface for immune surveillance of viruses and tumors. Whereas key steps of MHC-I assembly and its acquisition of peptides in the ER are relatively well defined, little is known about how MHC-I molecules leave the ER for cell surface expression. Here, we show that ER export of human classical MHC-I molecules (HLA-A/-B/-C) is regulated by their C-terminal single amino acid, valine or alanine. These amino acids, conserved in nearly all known human MHC-I alleles, serve as the ER export signal by binding to the Sec23/24 complex, a structural component of coat protein complex II (COPII) vesicles that mediate ER-to-Golgi trafficking. Together, our results strongly suggest that ER export of human classical MHC-I molecules can occur via a receptor-mediated process dictated by a highly conserved ER export signal.

HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion.

SNARE-dependent membrane fusion requires the disassembly of cis-SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans-SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans-SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans-SNARE complex, like cis-SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans-, but not in the cis-, configuration. This selective HOPS preservation of trans-SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X-100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans-SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic.

Human cytomegalovirus UL18 utilizes US6 for evading the NK and T-cell responses.

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses.

Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones.

The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.