Sec13 promotes glycolysis by inhibiting Ubqln1 mediated Pgm1 ubiquitination in ALI.

Acute lung injury (ALI) is a severe lung damage characterized by acute hypoxemia, increased pulmonary vascular permeability, and inflammatory reactions. Despite current treatments, mortality from ALI remains high. This study found that Sec13 is highly expressed in ALI and regulates it by glycolysis and epithelial-mesenchymal transition (EMT). In an ALI mouse model and cell model, Sec13 expression increased, accompanied by enhanced glycolysis, EMT, and inflammation. Sec13 knockdown suppressed these effects, alleviating ALI. Sec13 forms a protein complex with Pgm1, an enzyme regulating glucose-6-phosphate (G6P) production, and Ubqln1, an ubiquitin ligase. Sec13 inhibits Ubqln1-mediated Pgm1 ubiquitination, thereby stabilizing Pgm1. In ALI, Pgm1 binding to Sec13 increased but binding to Ubqln1 decreased. Sec13 knockdown decreased lactate, G6P, EMT markers, and inflammatory cytokines. Pgm1 knockdown produced similar effects. Ubqln1 overexpression suppressed inflammation but decreased Pgm1 expression. In conclusion, Sec13 plays a key role in ALI by inhibiting Ubqln1-mediated Pgm1 ubiquitination, affecting glycolysis and EMT. Sec13 and Pgm1 may be new targets for treating ALI.

"Off-pore" nucleoporins relocalize heterochromatic breaks through phase separation.

Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and "safe" homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear. Here, we show that Nup98 nucleoporin is recruited to heterochromatic repair sites before relocalization through Sec13 or Nup88 nucleoporins, and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing a novel "off-pore" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote.

SPOP negatively regulates mTORC1 activity by ubiquitinating Sec13.

The mammalian target of rapamycin complex1 (mTORC1) can response to amino acid to regulate metabolism and cell growth. GATOR2 act as important role in amino acid mediated mTORC1 signaling pathway by repressing GTPase activity (GAP) of GATOR1. However, it is still unclear how GATOR2 regulates mTORC1 signaling pathway. Here, we found that K63-ubiquitination of Sce13, one component of GATOR2, suppresses the mTORC1 activity by lessening the inter-interaction of GATOR2. Mechanistically, the ubiquitination of Sec13 was mediated by SPOP. Subsequently, the ubiquitination of Sec13 attenuated its interaction with the other component of GATOR2, thus suppressing the activity of mTORC1. Importantly, the deficiency of SPOP promoted the faster proliferation and migration of breast cancer cells, which was attenuated by knocking down of Sec13. Therefore, SPOP can act as a tumor suppressor gene by negatively regulating mTORC1 signaling pathway.

The ER-Golgi transport of influenza virus through NS1-Sec13 association during virus replication.

IMPORTANCE: Influenza A virus is a respiratory virus that can cause complications such as acute bronchitis and secondary bacterial pneumonia. Drug therapies and vaccines are available against influenza, albeit limited by drug resistance and the non-universal vaccine administration. Hence there is a need for host-targeted therapies against influenza to provide an effective alternative therapeutic target. Sec13 was identified as a novel host interactor of influenza. Endoplasmic reticulum-to-Golgi transport is an important pathway of influenza virus replication and viral export. Specifically, Sec13 has a functional role in influenza replication and virulence.

Arabidopsis SEC13B Interacts with Suppressor of Frigida 4 to Repress Flowering.

SECRETORY13 (SEC13) is an essential member of the coat protein complex II (COPII), which was reported to mediate vesicular-specific transport from the endoplasmic reticulum (ER) to the Golgi apparatus and plays a crucial role in early secretory pathways. In Arabidopsis, there are two homologous proteins of SEC13: SEC13A and SEC13B. SUPPRESSOR OF FRIGIDA 4 (SUF4) encodes a C2H2-type zinc finger protein that inhibits flowering by transcriptionally activating the FLOWERING LOCUS C (FLC) through the FRIGIDA (FRI) pathway in Arabidopsis. However, it remains unclear whether SEC13 proteins are involved in Arabidopsis flowering. In this study, we first identified that the sec13b mutant exhibited early flowering under both long-day and short-day conditions. Quantitative real-time PCR (qRT-PCR) analysis showed that both SEC13A and SEC13B were expressed in all the checked tissues, and transient expression assays indicated that SEC13A and SEC13B were localized not only in the ER but also in the nucleus. Then, we identified that SEC13A and SEC13B could interact with SUF4 in vitro and in vivo. Interestingly, both sec13b and suf4 single mutants flowered earlier than the wild type (Col-0), whereas the sec13b suf4 double mutant flowered even earlier than all the others. In addition, the expression of flowering inhibitor FLC was down-regulated, and the expressions of flowering activator FLOWERING LOCUS T (FT), CONSTANS (CO), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) were up-regulated in sec13b, suf4, and sec13b suf4 mutants, compared with Col-0. Taken together, our results indicated that SEC13B interacted with SUF4, and they may co-regulate the same genes in flowering-regulation pathways. These results also suggested that the COPII component could function in flowering in Arabidopsis.

Obesity Modifies the Proteomic Profile of the Periodontal Ligament.

This study aimed to assess the obesity effects on the proteomic profile of the periodontal ligament of rats submitted to obesity induction by a high-fat diet. Eight Holtzman rats were divided into control (n = 3) and obese (n = 5) groups. The maxillae were histologically processed for laser capture microdissection of the periodontal ligament of the first maxillary molars. Peptide mixtures were analyzed by LC-MS/MS. A total of 1379 proteins were identified in all groups. Among them, 335 (24.30%) were exclusively detected in the obese group, while 129 (9.35%) proteins were uniquely found in the control group. Out of the 110 (7.98%) differentially abundant proteins, 10 were more abundant and 100 had decreased abundance in the obese group. A gene ontology analysis showed some proteins related to obesity in the "extracellular exosome" term among differentially identified proteins in the gene ontology cellular component terms Prelp, Sec13, and Sod2. These three proteins were upregulated in the obese group (p < 0.05), as shown by proteomic and immunohistochemistry analyses. In summary, our study presents novel evidence that the proteomic profile of the periodontal ligament is altered in experimental obesity induction, providing a list of differentially abundant proteins associated with obesity, which indicates that the periodontal ligament is responsive to obesity.

Cloning and characterization of the target protein subunit lst8 of rapamycin in Apostichopus japonicus.

Autophagy plays an important role in the immune defense systems of vertebrates through the interaction between the lethal with SEC13 protein 8 (lst8) and the mechanistic target of rapamycin. In the present study, a novel invertebrate lst8 homologue is identified from Apostichopus japonicus (designated as Ajlst8) via polymerase chain reaction. The full-length complementary DNA of Ajlst8 comprises a 5'-untranslated region (UTR) of 78 base pair (bp), a 3'-UTR of 479 bp, and a putative open reading frame of 951 bp; hence, 316 amino acids are encoded. Structural analysis shows that the deduced amino acid of Ajlst8 shares six typical WD40 domains (28 aa-248 aa). Spatial expression analysis indicates that Ajlst8 is ubiquitously expressed in all the examined tissues, with a larger magnitude in coelomocytes. Vibrio splendidus infection in vivo and lipopolysaccharide exposure in vitro can significantly upregulate the messenger RNA expression of Ajlst8 by 2.39-fold and 1.93-fold compared with the control group, respectively. LPS exposure could also significantly induced the protein level of Ajlst8 to 2.38-fold and the autophagy level was markedly increased by 3.08-fold under same condition. The RNA interference of Ajlst8 in primary coelomocytes also reduces the relative expression of autophagy with a 0.71-fold decrease in the ratio of LC3-II/LC3-I compared with that in the control group. These results indicate that Ajlst8 is a novel immune regulator that may be involved in the antibacterial response process of sea cucumber by regulating autophagy.

Sec13 is a positive regulator of VISA-mediated antiviral signaling.

Viral infection triggers the innate antiviral immune response that rapidly produces type I interferons in most cell types to combat viruses invading. Upon viral infection, the cytoplasmic RNA sensors RIG-I/MDA5 recognize viral RNA, and then RIG-I/MDA5 is transported to mitochondria interacting with VISA through the CARD domain. From there, VISA recruits downstream antiviral signaling pathways molecules, such as TRAFs and TBK1. Eventually, IRF3 is phosphorylated and type I IFNs are induced to fight as the first line of defense against viruses. However, it remains unclear how VISA acts as a scaffold to assemble the signalosome in RIG-I-mediated antiviral signaling. Here, we demonstrated Sec13 as a novel component that was involved in VISA-mediated antiviral signaling pathway. The co-immunoprecipitation assays showed that Sec13 specifically interacts with VISA. Overexpression of Sec13 increases VISA's aggregation and ubiquitination and significantly enhances the phosphorylation and dimerization of IRF3, facilitating the IFN-ß production. Conversely, the knockdown of Sec13 attenuates Sendai virus-induced and VISA-mediated IRF3 activation and the production of IFNß, thus weakens antiviral immune activity.

Unexpected ancient paralogs and an evolutionary model for the COPII coat complex.

The coat protein complex II (COPII) is responsible for the transport of protein cargoes from the Endoplasmic Reticulum (ER) to the Golgi apparatus. COPII has been functionally characterized extensively in vivo in humans and yeast. This complex shares components with the nuclear pore complex and the Seh1-Associated (SEA) complex, inextricably linking its evolution with that of the nuclear pore and other protocoatomer domain-containing complexes. Importantly, this is one of the last coat complexes to be examined from a comparative genomic and phylogenetic perspective. We use homology searching of eight components across 74 eukaryotic genomes, followed by phylogenetic analyses, to assess both the distribution of the COPII components across eukaryote diversity and to assess its evolutionary history. We report that Sec12, but not Sed4 was present in the Last Eukaryotic Common Ancestor along with Sec16, Sar1, Sec13, Sec31, Sec23, and Sec24. We identify a previously undetected paralog of Sec23 that, at least, predates the archaeplastid clade. We also describe three Sec24 paralogs likely present in the Last Eukaryotic Common Ancestor, including one newly detected that was anciently present but lost from both opisthokonts and excavates. Altogether, we report previously undescribed complexity of the COPII coat in the ancient eukaryotic ancestor and speculate on models for the evolution, not only of the complex, but its relationship to other protocoatomer-derived complexes.

The nuclear pore complex function of Sec13 protein is required for cell survival during retinal development.

Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13(sq198) mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13(sq198), suggesting that the digestive organ defects observed in sec13(sq198) are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13(sq198) failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13(sq198). Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13(sq198). Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis.

A role for Raptor phosphorylation in the mechanical activation of mTOR signaling.

The activation of mTOR signaling is necessary for mechanically-induced changes in skeletal muscle mass, but the mechanisms that regulate the mechanical activation of mTOR signaling remain poorly defined. In this study, we set out to determine if changes in the phosphorylation of Raptor contribute to the mechanical activation of mTOR. To accomplish this goal, mouse skeletal muscles were subjected to mechanical stimulation via a bout of eccentric contractions (EC). Using mass spectrometry and Western blot analysis, we found that ECs induced an increase in Raptor S696, T706, and S863 phosphorylation, and this effect was not inhibited by rapamycin. This observation suggested that changes in Raptor phosphorylation might be an upstream event in the pathway through which mechanical stimuli activate mTOR. To test this, we employed a phospho-defective mutant of Raptor (S696A/T706A/S863A) and found that the EC-induced activation of mTOR signaling was significantly blunted in muscles expressing this mutant. Furthermore, mutation of the three phosphorylation sites altered the interactions of Raptor with PRAS40 and p70(S6k), and it also prevented the EC-induced dissociation of Raptor from p70(S6k). Combined, these results suggest that changes in the phosphorylation of Raptor play an important role in the pathway through which mechanical stimuli activate mTOR signaling.

SEACing the GAP that nEGOCiates TORC1 activation: evolutionary conservation of Rag GTPase regulation.

The target of rapamycin complex 1 (TORC1) regulates eukaryotic cell growth in response to a variety of input signals. In S. cerevisiae, amino acids activate TORC1 through the Rag guanosine triphosphatase (GTPase) heterodimer composed of Gtr1 and Gtr2 found together with Ego1 and Ego3 in the EGO complex (EGOC). The GTPase activity of Gtr1 is regulated by the SEA complex (SEAC). Specifically, SEACIT, a SEAC subcomplex containing Iml1, Npr2, and Npr3 functions as a GTPase activator (GAP) for Gtr1 to decrease the activity of TORC1 and, consequently, growth, after amino acid deprivation. Here, we present genetic epistasis data, which show that SEACAT, the other SEAC subcomplex, containing Seh1, Sea2-4, and Sec13, antagonizes the GAP function of SEACIT. Orthologs of EGOC (Ragulator), SEACIT (GATOR1), and SEACAT (GATOR2) are present in higher eukaryotes, highlighting the remarkable conservation, from yeast to man, of Rag GTPase and TORC1 regulation.

Huang-Lian-Jie-Du-Decotion induced protective autophagy against the injury of cerebral ischemia/reperfusion via MAPK-mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du-Decotion (HLJDD, Hwangryun-Hae-Dok-Decotion in Japan), an ancient antipyretic and detoxifying traditional Chinese medicine formula, was reported to have protective effect on ischemic stroke. AIM OF THE RESEARCH: To investigate the therapeutic effect of HLJDD on ischemic stroke and explore its mode of action. MATERIAL AND METHODS: A model of ischemic stroke in the rat was established after transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Rats were assigned randomly to groups of control, sham, transient ischemia/reperfusion (I/R), and three treatment groups by HLJDD at 2.5, 5.0, 10.0mg/kg. The neurological deficit, the cerebral infarct size, morphology abnormality, biochemical parameters were examined, and the levels of relevant proteins were determined by immunoblotting analysis to evaluate the protective effects of HLJDD on ischemic stroke and explore the underlying mechanism. RESULTS: Compared with I/R group, HLJDD significantly ameliorated neurological deficit and histopathology changes, decreased infarct area, and restored the levels of biochemical indicators including nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD and glutathione peroxidase (GSH-PX). HLJDD also notably elevated the levels of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and other autophagy related genes (Atgs), promoted the activation of extracellular signal-regulated kinases (ERK), protein kinase B (Akt), 3-phosphoinositide-dependent kinase (PDK1), and inhibited the activation of mammalian target of rapamycin (mTOR), c-Jun N-terminal protein kinases (JNK), p38, phosphatase and tensin homolog (PTEN). CONCLUSION: HLJDD showed neuroprotective effects on ischemic stroke, at least in part to the induced protective autophagy via the regulation of mitogen-activated protein kinase (MAPK) signals. This Akt-independent protective autophagy is favorable in the treatment of stroke, avoiding unfavorable side-effects associated with the inactivation of Akt. The efficacy of HLJDD on ischemic stroke and its safety warranted by its long-term clinical use in traditional Chinese medicine favored further study to develop HLJDD as an effective therapeutic agent to treat ischemic stroke.

Sec13 induces genomic instability in U2OS cells

Sec13p has been known as an endoplasmic reticulum-Golgi transport protein. Recently, it has also been shown to be required for the formation of septation in the fission yeast Schizosaccharomyces pombe. In the present study, we focused on the role of a human homolog of Saccharomyces cerevisiae SEC13, Sec13 protein during mitosis in U2OS cells. We found that the expression of Sec13 was constant throughout the cell cycle, and localized to the kinetochores at metaphase during mitosis. By using green fluorescent protein technology, we observed that Sec13 is required for evasion of mitotic arrest in response to spindle damage, leading to G1-like phase and apoptotic cell death. In addition, cells expressing exogenous Sec13 showed giant nuclei compared to endogenous ones in the absence of nocodazole. These results demonstrate that Sec13 is involved in the regulation of the metaphase/anaphase transition and may be functionally associated with mitotic machinery to maintain genomic stability during mitosis.