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Biomedical subjects

Jun Qin

Publications and source records attributed to Jun Qin.

At least 19 recordsLinked to original sources
PubMed2026

Nrsn1-Smarcc1 Coupling Regulates Neural Stem Cell Differentiation and Chronic-phase Recovery After Ischemic Stroke.

Stroke remains a leading cause of long-term neurological disability worldwide, largely due to irreversible neuronal loss and the limited regenerative capacity of the adult mammalian brain. Neural stem cells (NSCs) in the adult brain possess the potential to generate new neurons after injury, yet the molecular mechanisms regulating their neuronal differentiation following ischemic insult remain incompletely understood. Here, integrating single-cell multi-omics analyses with spatial transcriptomics, we systematically delineated cell type-specific spatiotemporal dynamics in the striatum of a mouse model of ischemia-reperfusion injury. We identified Neurensin 1 (Nrsn1) as a gene markedly upregulated during NSC-derived neuronal differentiation in the recovery phase. Mechanistically, Foxa2 directly activates Nrsn1 transcription, whereas Nrsn1 promotes neuronal differentiation by facilitating the nuclear translocation of the chromatin-remodeling factor Smarcc1 in vitro. In vivo, both endogenous NSCs and transplanted NSCs overexpressing Nrsn1 significantly enhanced neuronal regeneration and improved functional recovery in mice subjected to middle cerebral artery occlusion and reperfusion (MCAO/R). Collectively, these findings identify Nrsn1 as a key regulator of NSC neuronal differentiation and uncover a Nrsn1-Smarcc1 coupling mechanism that promotes neural regeneration after ischemic brain injury, highlighting a potential molecular target for strategies aimed at enhancing post-stroke recovery.

Foxa2↗
PubMed2005

ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor.

Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.

Amino Acid Motifs↗
PubMed2005

Molecular dissection of PINCH-1 reveals a mechanism of coupling and uncoupling of cell shape modulation and survival.

How cells couple and uncouple regulation of cellular processes such as shape change and survival is an important question in molecular cell biology. PINCH-1, a widely expressed protein consisting of five LIM domains and a C-terminal tail, is an essential focal adhesion protein with multiple functions including regulation of the integrin-linked kinase (ILK) level, cell shape, and survival signaling. We show here that the LIM1-mediated interaction with ILK regulates all these three processes. By contrast, the LIM4-mediated interaction with Nck-2, which regulates cell morphology and migration, is not required for the control of the ILK level and survival. Remarkably, a short 15-residue tail C-terminal to LIM5 is required for both cell shape modulation and survival, albeit it is not required for the control of the ILK level. The C-terminal tail not only regulates PINCH-1 localization to focal adhesions but also functions after it localizes there. These findings suggest that PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but yet distinct domain interactions.

Adaptor Proteins, Signal Transducing↗
PubMed2005

VEGF-integrin interplay controls tumor growth and vascularization.

Cross-talk between the major angiogenic growth factor, VEGF, and integrin cell adhesion receptors has emerged recently as a critical factor in the regulation of angiogenesis and tumor development. However, the molecular mechanisms and consequences of this intercommunication remain unclear. Here, we define a mechanism whereby integrin alpha v beta3, through activation, clustering, and signaling by means of p66 Shc (Src homology 2 domain containing), regulates the production of VEGF in tumor cells expressing this integrin. Tumors with "activatable" but not "inactive" beta3 integrin secrete high levels of VEGF, which in turn promotes extensive neovascularization and augments tumor growth in vivo. This stimulation of VEGF expression depends upon the ability of alpha v beta3 integrin to cluster and promote phosphorylation of p66 Shc. These observations identify a link between beta3 integrins and VEGF in tumor growth and angiogenesis and, therefore, may influence anti-integrin as well as anti-VEGF therapeutic strategies.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Structure of an ultraweak protein-protein complex and its crucial role in regulation of cell morphology and motility.

Weak protein-protein interactions (PPIs) (K(D) > 10(-6) M) are critical determinants of many biological processes. However, in contrast to a large growing number of well-characterized, strong PPIs, the weak PPIs, especially those with K(D) > 10(-4) M, are poorly explored. Genome wide, there exist few 3D structures of weak PPIs with K(D) > 10(-4) M, and none with K(D) > 10(-3) M. Here, we report the NMR structure of an extremely weak focal adhesion complex (K(D) approximately 3 x 10(-3) M) between Nck-2 SH3 domain and PINCH-1 LIM4 domain. The structure exhibits a remarkably small and polar interface with distinct binding modes for both SH3 and LIM domains. Such an interface suggests a transient Nck-2/PINCH-1 association process that may trigger rapid focal adhesion turnover during integrin signaling. Genetic rescue experiments demonstrate that this interface is indeed involved in mediating cell shape change and migration. Together, the data provide a molecular basis for an ultraweak PPI in regulating focal adhesion dynamics during integrin signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks.

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNA-PKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.

Amino Acid Sequence↗
PubMed2005

Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA.

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

ATP-Binding Cassette Transporters↗
PubMed2005

[Effects of paclitaxel combined with bone marrow stromal stem cells implantation on vascular endothelial repair and smooth muscle cells growth in vitro].

OBJECTIVE: To investigate the effects of paclitaxel combined with bone marrow stromal stem cells (MSCs) implantation on inhibiting the smooth muscle cells (SMCs) growth and promoting endothelial repair by developing an endothelial repair model in vitro. METHODS: In a cell coculture system, rabbit endothelial cells (ECs) and human MSCs were seeded in the lower chamber and rabbit SMCs were seeded in the upper chamber. 3H-TdR incorporation and PCNA protein expression were used to evaluate SMCs proliferation at the 10th day after paclitaxel application (1, 10, 100 nmol/L; 20 min). Fluorescence immunocytochemistry was employed to observe the Flk-1 and vWF protein expression on MSCs. RESULTS: The SMCs 3H-TdR incorporation of the MSCs implant group was significantly lower than that of the proliferative ECs group (1 nmol/L: 12 265 +/- 991 vs. 14 505 +/- 1013 cpm/well; 10 nmol/L: 8401 +/- 783 vs. 10 511 +/- 934 cpm/well; 100 nmol/L: 5880 +/- 569 vs. 7457 +/- 768 cpm/well, n = 6, P < 0.05), but higher than that of the confluent ECs group (1 nmol/L: 12 265 +/- 991 vs. 8671 +/- 642 cpm/well; 10 nmol/L: 8401 +/- 783 vs. 6175 +/- 743 cpm/well; 100 nmol/L: 5880 +/- 569 vs. 4423 +/- 406 cpm/well, n = 6, P < 0.05). The expression of SMCs PCNA protein in MSCs implant group was lower than that of the proliferative ECs group (1 nmol/L: 0.92 +/- 0.06 vs. 1.15 +/- 0.07; 10 nmol/L: 0.97 +/- 0.07 vs. 1.07 +/- 0.08; 100 nmol/L: 0.91 +/- 0.05 vs. 1.18 +/- 0.11, n = 6, P < 0.05), but higher than that of the confluent ECs group (1 nmol/L: 0.92 +/- 0.06 vs. 0.74 +/- 0.07; 10 nmol/L: 0.97 +/- 0.07 vs. 0.78 +/- 0.06; 100 nmol/L: 0.91 +/- 0.05 vs. 0.71 +/- 0.05, n = 6, P < 0.05). The MSCs did not express vWF or Flk-1 protein before coculture. Although none cell expressed vWF, some of the MSCs began to express Flk-1 protein after cocultured with mature ECs for 10 days. CONCLUSION: MSCs implantation can partly inhibit the delayed SMCs proliferation. The MSCs cocultured with paclitaxel-treated mature ECs have the ability to differentiate into ECs.

Bone Marrow Cells↗
PubMed2004

Identification and characterization of a novel Drosophila melanogaster glutathione S-transferase-containing FLYWCH zinc finger protein.

Glutathione SH-transferase (GST) is a 25-kDa protein and a member of a large family that plays a critical role in the cellular homeostasis of all organisms. In this report, we describe a novel GST-containing protein identified and cloned from Drosophila. This 1045 amino acid protein possesses a zinc finger domain with a tandem array of four FLYWCH zinc finger motifs at its N-terminus and a C-terminal domain that shares a 46% homology with GST. The gene maps to chromosome 3 at position 84C6. Further characterization of this protein shows that it localizes to the cytoplasm of fly cells and is expressed through all stages of fly embryonic development. It binds to glutathione-S agarose beads in vitro. These results indicate that this new protein belongs to the GST family, thus named a Drosophila GST-containing FLYWCH zinc finger protein (dGFZF).

Amino Acid Sequence↗
PubMed2004

Identification of a novel BRMS1-homologue protein p40 as a component of the mSin3A/p33(ING1b)/HDAC1 deacetylase complex.

Repression of gene transcription is mediated by histone deacetylases containing repressor-co-repressor complexes, which are recruited to promoters of target genes via interactions with sequence-specific transcription factors. The mammalian Sin3A co-repressor complex contains a core of at least seven proteins including the pRb-interacting protein RBP1 and a putative tumor suppressor p33(ING1b). By biochemical purification and mass spectrometry, we have identified a novel component p40 from this complex. p40 bears homology to both yeast Sds3, a component of yeast histone deacetylase complexes, and its mammalian homologue mSds3. The p40-associated complex purified from human cells shows a strong histone deacetylase activity. When tethered to a Gal-DNA binding domain, the Gal-p40 is able to significantly repress transcription of a Gal-luciferase promoter. Interestingly, database analysis reveals that p40 is also highly homologous to BRMS1, a breast carcinoma metastasis suppressor, and overexpression of p40 in human cells can significantly inhibit cell growth. Thus, our data indicate that p40 may be critically involved in transcription repression of cell growth-associated gene expression by recruiting the HDAC1 deacetylase complex.

Amino Acid Sequence↗
PubMed2004

Selective phosphorylations of the SRC-3/AIB1 coactivator integrate genomic reponses to multiple cellular signaling pathways.

Although several lines of evidence have indicated that the activity of SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 could be regulated by phosphorylation, an important question remained as to how different signaling pathways can act through limiting concentrations of the same SRC-3 molecule to exert different physiological functions. Herein, we report the successful identification of six functional in vivo SRC-3 phosphorylation sites. Interestingly, all phosphorylation sites are required for coactivation of estrogen and androgen receptors, but not all sites are required for coactivation of NF-kappaB. Different combinations of site-specific phosphorylations of SRC-3 are required for induction of IL-6 gene expression by TNF-alpha as compared to oncogenic transformation of MEFs. Mechanisms of pathway selectivity involve protein-protein interactions of differentially phosphorylated SRC-3 with downstream transcriptional activators and coactivators. Our results uncovered an additional level of transcriptional regulation whereby specific modulations of SRC-3 phosphorylation allow this coactivator to function as a regulatable integrator for diverse signaling pathways in cells.

Acetyltransferases↗
PubMed2004

Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins.

In mammalian cells, telomere-binding proteins TRF1 and TRF2 play crucial roles in telomere biology. They interact with several other telomere regulators including TIN2, PTOP, POT1, and RAP1 to ensure proper maintenance of telomeres. TRF1 and TRF2 are believed to exert distinct functions. TRF1 forms a complex with TIN2, PTOP, and POT1 and regulates telomere length, whereas TRF2 mediates t-loop formation and end protection. However, whether cross-talk occurs between the TRF1 and TRF2 complexes and how the signals from these complexes are integrated for telomere maintenance remain to be elucidated. Through gel filtration and co-immunoprecipitation experiments, we found that TRF1 and TRF2 are in fact subunits of a telomere-associated high molecular weight complex (telosome) that also contains POT1, PTOP, RAP1, and TIN2. We demonstrated that the TRF1-interacting protein TIN2 binds TRF2 directly and in vivo, thereby bridging TRF2 to TRF1. Consistent with this multi-protein telosome model, stripping TRF1 off the telomeres by expressing tankyrase reduced telomere recruitment of not only TIN2 but also TRF2. These results help to unify previous observations and suggest that telomere maintenance depends on the multi-subunit telosome.

Blotting, Western↗
PubMed2004

Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase.

Turnover of cyclin E is controlled by SCF(Fbw7). Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7alpha and -gamma are nuclear and the beta-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay. Cyclin E is phosphorylated on Thr(62), Ser(88), Ser(372), Thr(380), and Ser(384) in vivo. To examine the roles of phosphorylation in cyclin E turnover, a series of alanine point mutations in each of these sites were analyzed for Fbw7-driven degradation. As expected, mutation of the previously characterized residue Thr(380) to alanine led to profound defects of cyclin E turnover, and largely abolished association with Fbw7. Mutation of Thr(62) to alanine led to a dramatic reduction in the extent of Thr(380) phosphorylation, suggesting an indirect effect of this mutation on cyclin E turnover. Nevertheless, phosphopeptides centered at Thr(62) associated with Fbw7, and residual binding of cyclin E(T380A) to Fbw7 was abolished upon mutation of Thr(62), suggesting a minor role for this residue in direct association with Fbw7. Mutation of Ser(384) to alanine also rendered cyclin E resistant to degradation by Fbw7, with the largest effects being observed with Fbw7beta. Cyclin E(S384A) associated more weakly with Fbw7alpha and -beta isoforms but was not defective in Thr(380) phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin E(S384A) in the nucleus, which may contribute to the inability of cytoplasmic Fbw7beta to promote turnover of this cyclin E mutant protein.

Amino Acid Motifs↗
PubMed2004

Protein phosphatase 4 interacts with and down-regulates insulin receptor substrate 4 following tumor necrosis factor-alpha stimulation.

Protein phosphatase 4 (PP4; also named PPX or PPP4) is a PP2A-related protein serine/threonine phosphatase with important roles in a variety of cellular processes such as microtubule growth/organization, apoptosis, tumor necrosis factor (TNF)-alpha signaling, and activation of c-Jun N-terminal kinase and NF-kappaB. To further investigate the cellular functions of PP4, we isolated and identified PP4-interacting proteins using a proteomic approach. We found that insulin receptor substrate 4 (IRS-4) interacted with PP4 and that this interaction was enhanced following TNF-alpha stimulation. We also found that PP4, but not PP2A, down-regulated IRS-4 in a phosphatase activity-dependent manner. Pulse-chase analysis revealed that PP4 decreased the half-life of IRS-4 from 4 to 1 h. Moreover, we found that TNF-alpha stimulated a PP4-dependent degradation of IRS-4, as indicated by the blockage of the degradation by a potent PP4 inhibitor (okadaic acid) and a phosphatase-dead PP4 mutant (PP4-RL). Taken together, our studies indicate that IRS-4 is subject to regulation by TNF-alpha and that PP4 mediates TNF-alpha-induced degradation of IRS-4.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Prospero-related homeobox (Prox1) is a corepressor of human liver receptor homolog-1 and suppresses the transcription of the cholesterol 7-alpha-hydroxylase gene.

Cholesterol 7-alpha-hydroxylase (CYP7A1) catalyzes a rate-limiting step in bile acid synthesis in liver, and its gene transcription is under complex regulation by multiple nuclear receptors in response to bile acids, cholesterol derivatives, and hormones. The liver receptor homolog-1 (LRH-1), a member of the fushi tarazu factor 1 subfamily of nuclear receptors, has emerged as an essential regulator for the expression of cyp7a1. In this report, we demonstrate Prox1, a prospero-related homeobox transcription factor, identified through a yeast two-hybrid screening, can directly interact with human LRH-1 (hLRH-1) and suppresses hLRH-1-mediated transcriptional activation of human cyp7a1 gene. Biochemical analysis demonstrates that Prox1 interacts with both the ligand binding domain (LBD) and the DNA binding domain (DBD) of hLRH-1. An LRKLL motif in Prox1 is important for the interaction with the LBD but not the DBD of hLRH-1. In hLRH-1 LBD, helices 2 and 10 are essential for Prox1 recruitment. The suppression by Prox1 on the transcriptional activity of hLRH-1 can be mediated through its interaction with the LBD or the DBD of hLRH-1. Gel shift assays reveal that Prox1 impairs the binding of hLRH-1 to the promoter of human cyp7a1 gene.

Amino Acid Sequence↗
PubMed2004

PTOP interacts with POT1 and regulates its localization to telomeres.

Telomere maintenance has been implicated in cancer and ageing, and requires cooperation between a multitude of telomeric factors, including telomerase, TRF1, TRF2, RAP1, TIN2, Tankyrase, PINX1 and POT1 (refs 1-12). POT1 belongs to a family of oligonucleotide-binding (OB)-fold-containing proteins that include Oxytricha nova TEBP, Cdc13, and spPot1, which specifically recognize telomeric single-stranded DNA (ssDNA). In human cells, the loading of POT1 to telomeric ssDNA controls telomerase-mediated telomere elongation. Surprisingly, a human POT1 mutant lacking an OB fold is still recruited to telomeres. However, the exact mechanism by which this recruitment occurs remains unclear. Here we identify a novel telomere protein, PTOP, which interacts with both POT1 and TIN2. PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres. Inhibition of PTOP by RNA interference (RNAi) or disruption of the PTOP-POT1 interaction hindered the localization of POT1 to telomeres. Furthermore, expression of the respective interaction domains on PTOP and POT1 alone extended telomere length in human cells. Therefore, PTOP heterodimerizes with POT1 and regulates POT1 telomeric recruitment and telomere length.

Active Transport, Cell Nucleus↗
PubMed2004

The human Rap1 protein complex and modulation of telomere length.

Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells. Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2. To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis. In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex. We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2. PARP1, however, most likely interacted with hRap1 through TRF2. Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres. In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres. Deletion of a novel linker domain of hRap1 (residues 199-223), however, abolished the dominant negative effect of hRap1 overexpression. These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length.

Antigens, Nuclear↗