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Chao Zhang

Publications and source records attributed to Chao Zhang.

At least 55 records · Page 3Linked to original sources
PubMed2005

Chemical genetic engineering of G protein-coupled receptor kinase 2.

G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.

Adenosine Triphosphate↗
PubMed2005

Structural bioinformatics-based design of selective, irreversible kinase inhibitors.

The active sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. We used a structural bioinformatics approach to identify two selectivity filters, a threonine and a cysteine, at defined positions in the active site of p90 ribosomal protein S6 kinase (RSK). A fluoromethylketone inhibitor, designed to exploit both selectivity filters, potently and selectively inactivated RSK1 and RSK2 in mammalian cells. Kinases with only one selectivity filter were resistant to the inhibitor, yet they became sensitized after genetic introduction of the second selectivity filter. Thus, two amino acids that distinguish RSK from other protein kinases are sufficient to confer inhibitor sensitivity.

Amino Acid Sequence↗
PubMed2005

The crystal structures of human steroidogenic factor-1 and liver receptor homologue-1.

Steroidogenic factor-1 (SF-1) and liver receptor homologue-1 (LRH-1) belong to the fushi tarazu factor 1 subfamily of nuclear receptors. SF-1 is an essential factor for sex determination during development and regulates adrenal and gonadal steroidogenesis in the adult, whereas LRH-1 is a critical factor for development of endodermal tissues and regulates cholesterol and bile acid homeostasis. Regulatory ligands are unknown for SF-1 and LRH-1. A reported mouse LRH-1 structure revealed an empty pocket in a region commonly occupied by ligands in the structures of other nuclear receptors, and pocket-filling mutations did not alter the constitutive activity observed. Here we report the crystal structures of the putative ligand-binding domains of human SF-1 at 2.1-A resolution and human LRH-1 at 2.5-A resolution. Both structures bind a coactivator-derived peptide at the canonical activation-function surface, thus adopting the transcriptionally activating conformation. In human LRH-1, coactivator peptide binding also occurs to a second site. We discovered in both structures a phospholipid molecule bound in a pocket of the putative ligand-binding domain. MS analysis of the protein samples used for crystallization indicated that the two proteins associate with a range of phospholipids. Mutations of the pocket-lining residues reduced the transcriptional activities of SF-1 and LRH-1 in mammalian cell transfection assays without affecting their expression levels. These results suggest that human SF-1 and LRH-1 may be ligand-binding receptors, although it remains to be seen if phospholipids or possibly other molecules regulate SF-1 or LRH-1 under physiological conditions.

Amino Acid Sequence↗
PubMed2005

Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma.

Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin's lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra beta-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.

Adenylyl Imidodiphosphate↗
PubMed2005

A chemical-genetic approach to studying neurotrophin signaling.

Trk tyrosine kinases are receptors for members of the neurotrophin family and are crucial for growth and survival of specific populations of neurons. Yet, the functions of neurotrophin-Trk signaling in postnatal development as well as maintenance and plasticity of the adult nervous system are less clear. We report here the generation of mice harboring Trk knockin alleles that allow for pharmacological control of Trk kinase activity. Nanomolar concentrations of either 1NMPP1 or 1NaPP1, derivatives of the general kinase inhibitor PP1, inhibit NGF and BDNF signaling in TrkA(F592A) and TrkB(F616A) neurons, respectively, while no such Trk inhibition is observed in wild-type neurons. Moreover, oral administration of 1NMPP1 leads to specific inhibition of TrkA(F592A), TrkB(F616A), and TrkC(F167A) signaling in vivo. Thus, Trk knockin mice provide valuable tools for selective, rapid, and reversible inhibition of neurotrophin signaling in vitro and in vivo.

Amino Acid Sequence↗
PubMed2005

An unbiased cell morphology-based screen for new, biologically active small molecules.

We have implemented an unbiased cell morphology-based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase-inhibitor scaffolds. Cellular phenotypes induced by each compound were quantified by multivariate statistical analysis of the morphology, staining intensity, and spatial attributes of the cellular nuclei, microtubules, and Golgi compartments. Principal component analysis was used to identify inhibitors of cellular components not targeted by known protein kinase inhibitors. Here we focus on a hydroxyl-substituted analog (hydroxy-PP) of the known Src-family kinase inhibitor PP2 because it induced cell-specific morphological features distinct from all known kinase inhibitors in the collection. We used affinity purification to identify a target of hydroxy-PP, carbonyl reductase 1 (CBR1), a short-chain dehydrogenase-reductase. We solved the X-ray crystal structure of the CBR1/hydroxy-PP complex to 1.24 A resolution. Structure-based design of more potent and selective CBR1 inhibitors provided probes for analyzing the biological function of CBR1 in A549 cells. These studies revealed a previously unknown function for CBR1 in serum-withdrawal-induced apoptosis. Further studies indicate CBR1 inhibitors may enhance the effectiveness of anticancer anthracyclines. Morphology-based screening of diverse cancer cell types has provided a method for discovering potent new small-molecule probes for cell biological studies and anticancer drug candidates.

Alcohol Oxidoreductases↗
PubMed2005

FastContact: rapid estimate of contact and binding free energies.

UNLABELLED: Interaction free energies are crucial for analyzing binding propensities in proteins. Although the problem of computing binding free energies remains open, approximate estimates have become very useful for filtering potential binding complexes. We report on the implementation of a fast computational estimate of the binding free energy based on a statistically determined desolvation contact potential and Coulomb electrostatics with a distance-dependent dielectric constant, and validated in the Critical Assessment of PRotein Interactions experiment. The application also reports residue contact free energies that rapidly highlight the hotspots of the interaction. AVAILABILITY: The program was written in Fortran. The executable and full documentation is freely available at http://structure.pitt.edu/software/FastContact

Algorithms↗
PubMed2005

Global mapping of the protein structure space and application in structure-based inference of protein function.

We have constructed a map of the "protein structure space" by using the pairwise structural similarity scores calculated for all nonredundant protein structures determined experimentally. As expected, proteins with similar structures clustered together in the map and the overall distribution of structural classes of this map followed closely that of the map of the "protein fold space" we have reported previously. Consequently, proteins sharing similar molecular functions also were found to colocalize in the protein structure space map, pointing toward a previously undescribed scheme for structure-based functional inference for remote homologues based on the proximity in the map of the protein structure space. We found that this scheme consistently outperformed other predictions made by using either the raw scores or normalized Z-scores of pairwise DALI structure alignment.

Proteins↗
PubMed2005

A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design.

Cyclic nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes that regulate a variety of cellular processes. We describe a family of potent PDE4 inhibitors discovered using an efficient method for scaffold-based drug design. This method involves an iterative approach starting with low-affinity screening of compounds followed by high-throughput cocrystallography to reveal the molecular basis underlying the activity of the newly identified compounds. Through detailed structural analysis of the interaction of the initially discovered pyrazole carboxylic ester scaffold with PDE4D using X-ray crystallography, we identified three sites of chemical substitution and designed small selective libraries of scaffold derivatives with modifications at these sites. A 4,000-fold increase in the potency of this PDE4 inhibitor was achieved after only two rounds of chemical synthesis and the structural analysis of seven pyrazole derivatives bound to PDE4B or PDE4D, revealing the robustness of this approach for identifying new inhibitors that can be further developed into drug candidates.

Binding Sites↗
PubMed2005

Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis.

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.

Amino Acid Sequence↗
PubMed2005

A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases.

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

Amino Acid Substitution↗
PubMed2005

[Tissue engineered bone regeneration of periosteal cells using recombinant human bone morphogenetic protein 2 induce].

OBJECTIVE: To investigate bone regeneration of the cell-biomaterial complex using strategies of tissue engineering based on cells. METHODS: Hydroxyapatite/collagen (HAC) sandwich composite was produced to mimic the natural extracellular matrix of bone, with type I collagen serving as a template for apatite formation. A three-dimensional poly-porous scaffold was developed by mixing HAC with poly(L-lactic acid) (PLA) using a thermally induced phase separation technique (TIPS). The rabbit periosteal cells were treated with 500 ng/ml of recombinant human bone morphogenetic protein 2 (rhBMP-2), followed by seeded into pre-wet HAC-PLA scaffolds. Eighteen 3-month nude mice were implanted subcutaneously cell suspension (group A, n = 6), simple HAC-PLA scaffold (group B, n = 6) and cell-biomaterial complex (group C, n = 6) respectively. RESULTS: Using type I collagen to template mineralization of calcium and phosphate in solution, we get HAC sandwich composite, mimicking the natural bone both in composition and microstructure. The three dimensional HAC-PLA scaffold synthesized by TIPS had high porosity up to 90%, with pore size ranging from 50 microm to 300 microm. SEM examination proved that the scaffold supported the adhesion and proliferation of the periosteal cells. Histology results showed new bone formation 8 weeks after implantation in group C. The surface of group A was smooth without neoplasma. Fibrous tissue invasion occurred in group B and no bone and cartilage formations were observed. CONCLUSION: The constructed tissue engineering bone has emerged as another promising alternative for bone repair.

Animals↗
PubMed2005

[The expression of ICAM-1 in laryngeal carcinoma and it's significance].

OBJECTIVE: To study the expression of ICAM-1 in laryngeal carcinoma and it's significance. METHOD: ICAM-1 were detected in 78 cases laryngeal carcinoma, 30 cases dysplasia and 5 cases normal by SP immunohistochemistry. RESULT: ICAM-1 can be expressed on the lymphocytes of patients with laryngeal carcinoma, but not cells of normal, dysplasia and cancer. The first and second stages laryngeal carcinoma had stronger ICAM-1 expression than those of the third and fourth stages (P < 0.05). ICAM-1 expression in laryngeal carcinoma with cervical metastasis was higher than that without cervical metastasis (P < 0.05). Three and five years survival rate in patients with higher ICAM-1 expression were higher than those of patients with lower ICAM-1 expression (P < 0.05). CONCLUSION: These results implicate that the development, metastasis and prognosis of laryngeal carcinoma may be correlated to ICAM-1 expression of lymphocyte.

Adult↗
PubMed2005

[Clinical management and prognostic analysis of primary peritoneal neoplasms].

OBJECTIVE: To investigate the clinical management strategies and prognostic factors of primary peritoneal neoplasms. METHODS: We retrospectively reviewed the clinical and pathological records of 24 cases with primary peritoneal neoplasms treated in the People's Hospital, Peking University during May 1995 and April 2004. RESULTS: Among 24 cases, 15 patients were diagnosed as serous papillary adenocarcinoma (9 highly and intermediately differentiated, and 6 lowly differentiated), 6 as mixed epithelial carcinoma and 3 as mixed malignant Mullerian tumor (MMMT). All patients underwent cytoreductive surgery, 21 cases having, suboptimal debulking one. Then they received a platinum-based chemotherapy. Thirteen cases received paclitaxel + cisplatin (TP) and 9 received cisplatin + doxorubicin + cyclophosphamide (PAC) combination chemotherapy. The primary response reached 80% (complete response 55% and partial response 25%). The median survival of all patients was 42 months (95% CI = 22-62 months). Survival for patients with primary peritoneal serous papillary carcinoma (PPSPC), mixed epithelial carcinoma and MMMT was 44, 19 and 13 months respectively, with a significant difference between PPSPC and MMMT (P < 0.05). Patients receiving TP combination also exhibited longer survival than those receiving PAC regimen (mean survival 75 vs 28 months, P < 0.05). CONCLUSIONS: Patients with primary peritoneal neoplasms should be treated with appropriate cytoreductive surgery. A primary surgical protocol is bilateral salpingo-oophorectomy and omentectomy. Overestimating an optimal debulking surgery may have no benefit on the survival. TP combination therapy may bring longer survival than PAC regimen. Histopathologic types and chemotherapy regimens are the essential factors of the prognosis.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

[Temperature changes of patients with severe acute respiratory syndrome: a comparative study].

OBJECTIVE: To explore the patterns of temperature changes of patients with the severe acute respiratory syndrome (SARS) and the effect of glucocorticoid hormone on the temperature of these patients. METHODS: The clinical data of 94 SARS cases treated during the outbreak of SARS in South China in 2003 were collected for a retrospective review. According to different treatment regimens, the patients were divided into hormone group (n=35) and non-hormone group (n=59). The control groups consisted of 65 patients with interstitial pneumonia, 78 with bacterial pneumonia and 57 with upper respiratory tract infection. The changes in body temperature were compared between the SARS patients and those with other respiratory diseases and the effect of glucocorticoid hormone on controlling body temperature of the SARS patients was explored. RESULTS: The body temperature of patients with the 4 diseases all exhibited obvious reduction 7 days after hospitalization (P<0.001) with only subsequent mild fluctuation within the basically normal range. At each time point of measurement, the body temperature of SARS patients was significantly higher than that of patients with other diseases (P<0.03), with a fluctuation of 0.2 to 0.5 degrees C; and following a pattern of variation similar to those of the other diseases. Of the 4 time points of daily measurement, namely 6, 10, 14 and 18 o'clock, the temperature measured at 14 o'clock was significantly higher than those at the other 3 time points (P<0.001). Hormone therapy did not significantly affect the temperature of SARS patients (P=0.180), who had longer duration of high fever. CONCLUSION: SARS patients have higher body temperature and longer duration of high fever. Hormone therapy may not produce significant effect in controlling the temperature of SARS patients.

Adult↗
PubMed2005

[The effect of bone-related growth factors on the proliferation and differentiation of marrow mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the effect of dexamethasone, recombinant human fibroblast growth factor (rhFGF) and recombinant human bone morphogenetic protein 2 (rhBMP-2) on the proliferation and differentiation of marrow stromal stem cells (MSCs) for their further application in tissue engineering. METHODS: MSCs were isolated and cultured in vitro, and then exposed to different dose of dexamethasone (10(-8) mol/l, 10(-7) mol/L, 10(-6) mol/L), rhFGF (50 ng/ml, 200 ng/ml, 500 ng/ml) and rhBMP-2 (50 ng/ml, 500 ng/ml, 1000 ng/ml) respectively. The total protein and alkaline phosphatase (ALP) activity of each group was measured on 4th and 7th day. RESULTS: Exposure of MSCs with 10(-6) mol/L dexamethasone inhibited protein synthesis without obvious effects on ALP expression. The application of rhFGF significantly promoted cell proliferation but inhibited ALP activity. In comparison, ALP expression was significantly enhanced by treatment of rhBMP-2 at concentration of 500 ng/ml, 1000 ng/ml. CONCLUSION: The exposure of dexamethasone as well as rhBMP-2 to MSCs with an appropriate concentration promotes osteogenic expression without reverse effects on cell proliferation, which indicates the great potential value in cell-based strategy of bone tissue engineering.

Alkaline Phosphatase↗
PubMed2005

Selecting stable molecular targets for treatment and prevention of AIDS.

The common consideration in approaching protection against HIV, whether by a vaccine or therapeutic, is identification of suitable targets. Among the central criteria for suitability is target stability; i.e. resistance to mutation. In this paper we address the problem of stability, and develop methods for identifying stable targets. The targets that we focus on are structures formed by viral peptides and products of the class I major histocompatibility complex, the target of the immune system. The method mines the large databases of fully sequenced HIV genomes and MHC binding peptides, and takes account of human polymorphism to construct hundreds of subpopulation specific stable targets, each consisting of combinations of 3-5 complexes.

Acquired Immunodeficiency Syndrome↗