Keping Hu, Ph.D. hukeping @gmail.com
Keping Hu, Ph.D.
Email:
EDUCATION
Certificate. Principles of Clinical Pharmacology.
The Warren Magnuson Clinical Center of the National Institutes of Health, Bethesda, MD, USA, 2004
Ph.D., in Biochemistry
Research Institute of Pharmaceutical Chemistry, Beijing, China (with National Laboratory of Protein Engineering, Peking University, at Professor Bingang Ru’s lab), Beijing, China, July 1997
B.S., Biology
Jiangxi University, Nanchang, China, July 1985
PROFESSIONAL EXPERIENCE
Principal Investigator, 12/2009 to present
Institute of Medicinal Plant Development (IMPLAD)
Chinese Academy of Medical Sciences (CAMS)
Peking Union Medical College (PUMC), Beijing, China
Assistant Research Biochemist (Professor), Step II. 08/2007 to present
Molecular & Medical Pharmacology, UCLA School of Medicine, in
Dr. Yi Sun Lab.
Manager, gsk R&D China, Shanghai. 03/2008 to present
Set up stem cell lab in gsk R&D China, focused on functional study of Oct 4, Sox2 in stem cell. As a lab manager, I set up a state-of-the art stem cell research lab in gsk R&D China in 3 months.
Project 1)MeCP2 phosphorylation sites mapping and phosphor-state specific antibodies development
MeCP2 mutations cause Rett syndrome. I independently discovered MeCP2 is a phosphorylated protein. It was reported that MeCP2 phosphorylation plays critical role in MeCP2 function. I used one-step immunoprecipitation approach coupled with mass spectrometry, to map MeCP2 phosphorylation sites from HeLa cells, mouse and rat brain tissues, and developed 8 different phosphorylation antibodies to fit the sites in MeCP2. Presently, I am mapping MeCP2 phosphorylation site directly from human brain tissue both in normal and Rett patient, which will provide MeCP2 phosphorylation information to help clinical and basic research on Rett syndrome. Subsequently, I am going to develop antibodies to all newly mapped sites. Those tools could be great help to Rett syndrome research, and potentially to develop an approach to cure Rett syndrome, a neurodevelopmental disorder predominantly in girls.
2) Dnmt3a, a DNA methylation enzyme, protein complexes immunoprecipitation and characterization.
DNA methylation provides epgenetics traits in development. Dnmt3a plays a key role in regulating DNA methylation. To study Dnmt3a function, a Dnmt3a associated protein complex will be explored. I am going to purify this protein based on my previous experience on MeCP2, SirT1, and Smarc 1complezes. I will use Dnmt3a wild type and -/- ES cells developed in Dr. Yi Sun lab for this protein complex characterization.
RESEARCH ASSOCIATE, 07/05 to 08/07
Biomedical Research Department, A.I. duPont Hospital for Children, Wilmington, DE
PI: Dr. Carolyn Schanen
Project Rett Syndrome Protein MeCP2, Ser80 Phosphorylation Pathway and Its Dynamic Function in PC12 Cells
Scientific Summary:
Utilized NGF treatment to find kinase(s) for Ser80 phosphorylation in cells, because Ser80 is conserved in human, mouse and rat, phosphorylation of Ser80 could play a role in MeCP2 function in cells. I hope this research could get more understanding how MeCP2 mutation is linked to Rett Syndrome.
Techniques Employed:
Cell culture based NGF plus K252a, LY and PD, TrAk pathway drugs treatment. The most important approaches are immunoprecipitation(IP) and Western blot. Because NGF is so important in neurons development, I try to map phosphorylation site(s) specific under NGF treatment with the help of Mass Spectrometry. I already mapped the normal PC12 cells phosphorylation sites in this lab.
Additional Responsibility:
I made a major contribution to help Dr. Schanen to get NIH RO1 grant.
POSTDOC, 11/01 to 07/05
National Institute on Aging, Baltimore, MD
PI: Dr. Weidong Wang
Projects: 1. Human Life Span Factor Sir2 Complex, the Characterization and Function Studies.
Scientific Summary:
Sir2 is a very important life span factor in yeast, for example, a double copy of Sir2 gene expressed in daughter cells, which is significantly longer life span than the mother cells does. In yeast, scientists have purified a Sir2 complex. My main task was to find Sir2 complex in human. Utilizing immunoprecipitation (IP) and Western bolt purified human Sir2 protein complex from HeLa cells. I found Sir2 is a shuttle protein both existing in cytoplasm and unclear, while it is only existing in unclear in yeast. It means Sir2 function is evolutionally changed in human cells.
Techniques Employed:
Immunoprecipitation(IP), Western blot, silver staining and Coomassie staining, cell culture, immunoflurosence for Sir2 location, NMDA activity assay coupled with Mass Spectrometry identification Sir2 complex purification.
Projects: 2. Rett Syndrome Protein, MeCP2, Characterization and Function Studies
Scientific Summary:
Rett Syndrome (RTT) is a debilitating neurological disorder diagnosed almost exclusively in females. Children with RTT appear to develop normally until 6 to 18 months of age when they enter a period of regression, losing speech and motor skills. Most develop repetitive hand movements, irregular breathing patterns, seizures and extreme motor control problems. RTT leaves its victims profoundly disabled, requiring maximum assistance with every aspect of daily living. Historically, RTT as believed to affect 1 in 10,000 females. There is no cure.
The leading cause of RTT is sporadic mutations in a gene called MECP2, located on the X chromosome. Studies have shown that more then 95% of mutations originate from a mutated sperm. The MECP2 gene makes a protein, also called MeCP2, believed to play a pivotal role in silencing other genes. Scientists suspect that the inability to shut down specific genes causes the cascade of symptoms seen in RTT.
I tired to utilize the powerful single step immunoprecipitation(IP) approach developed at Dr. Weidong Wang’s lab to purify the endogenous MeCP2 from cells lines and brain tissue. Eventhrough there has been a MeCP2 complex published in a leading journal of Nature Genetics, which showed MeCP2 formed a complex with HDAC1 and Sin3A. I questioned this purification due to no mass spectrometry identification data yet. I tried to use commercial MeCP2 antibodies in my initial study, after all efforts failed to immunoprecipitation(IP), I started work on this project with the beginning develop MeCP2 antibody in my own. I used cDNA ->PCR->MeCP2 plasmid->bacteria overexpression->purification protein as immungen->immune rabbit->serum infinity putification->MeCP2 polyclone antibody. I used this antibody successfully IPed MeCP2 from human (HeLa), normal and seized mouse and rat brain tissues. Those immunoprecipitations all identified by mass spectrometry. Thus, I was first to immunoprecipitate endogenous MeCP2 complex. This MeCP2 antibody is working well in IP, Western bolt, and immunoflurosence. With commercial MeCP2 antibody, only Upstate can be only used in Western blot, and all others failed to work. That’s the reason Rett Syndrome Research Foundation (RSRF) is highly recommended our antibody in its web site (www.rsrf.org/researchers/4.3 ).
My major contribution in MeCP2 field is I make people rethink the MeCP2 function model they had, for example, I have strong evidence showing that MeCP2 forms a complex in its own, and does not stable associate with other proteins in human (HeLa cells), mouse (3T3 cells and brain tissues both normal and seized) and rat (PC12 cells and brain tissues both normal and seized). I got those data from IPs following silver and Commassie staining coupled with mass spectrometry identification. This part of research already published in Nature Genetics in September 2006.
I made another major contribution is I independently found MeCP2 phosphorylation. I mapped most phosphorylation sites of MeCP2 from human (HeLa), mouse and rat brain tissues. Part of this research was published in PNAS on March 24, 2009.
Techniques Employed:
PCR, DNA purification, plasmids transfection, bacteria overexpression target protein, and its purification, antibody affinity purification, Immunoprecipitation(IP), Western blot, silver staining and Coomassie staining, cell culture, cell synchronization, primary neuron cell culture and KCl treatment, immunoflurosence for MeCP2 or phosphorylation MeCP2 localization, protein Mass Spectrometry identification and phosphorylation mapping.
POSTDOC, 07/00 to11/01
UNC-Chapel Hill, Chapel Hill, NC
PI: Dr. Darryl Stafford
Project: 1. Characterized the Carboxylase and Reductase in the Toxin-ducts of Conus,
2. Biosynthesis and Chemical Purification of QS, a Carboxylase specific substrate.
Scientific Summary:
Carboxylase is an enzyme, which add a –COO group to other molecule and plays a very important role in blood system. It is confirmed that venom from Cone contains rich carboxylase. Dr. Stafford’s long term goal is to find reductase, which removes a -COO from other molecule. And I did another project: bacteria overexpressed a specific substrate QS, then chemical cut it via CNBr through chemicals purification.
Techniques Employed:
Carboxylase activity assay, PCR, DNA purification, DNA plasmid making, bacteria overpression target protein, CNBr cutting and chemical extract protein purification. HPLC purification.
POSTDOC, 09/97 to 01/00
National Laboratory of Biomembrane and Membrane Bioengineering
Peking University, Beijing, China
PI: Professor CaiHong Wu
Project: Structure and Physiological Function Studies of Conotoxin from Conus textile,
Scientific Summary:
Conotoxins, which are peptides consisting of 10 to 30 amino acid residues, typically have some disulfide bonds. Conotoxins have a variety of activities, most of which have not been explained closely yet. The number of conotoxins whose activities have been determined so far is five, and they are called the α-, δ-, κ-, μ-, and ω types. I finished manuscript and accepted by FEBS letter with the title: A new cysteine framework of conotoxin from Couns betulinus. This 27 amino acids new peptide provided a new framework for conotoxin family, and act as a K channel blockade. This new peptide has been got its sequence record in Swiss Port.
Techniques Employed:
Cones identification and classification in biological level, cone venom collection, G-25, G100, Super6 column, ion exchange column purification, HPLC purification. Protein vacuum-dry technique, Whole Cell-patch Clamp technique, peptide digest following Edman degradation for protein sequencing coupled with mass spectrometry identification. I also worked on using frog and cockroach as samples for characterization of both cone venom and purified conotoxin in physiology.
VISITING SCHOOLAR , 10/93 to 5/94
National Laboratory of Protein Engineering, Peking University, Beijing, China
PI: BingGen Ru
Project: Metallothionein Purification from the Extracts of Pig Liver
Scientific Summary:
Liver is a major target organ of cadmium (Cd) toxicity following acute and chronic exposure. Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding protein has been shown to play an important role in protection against acute Cd-induced liver injury.
Techniques Employed:
Protein purification from liver extracts coupled with size and ion column purification.
LECTURE, 09/87 to10/93
Jiangxi University, Nanchang, China
Teaching course: * Teaching General Microbiology and Immunology to undergraduate students.
* Directing General Microbiology & Immunology experiments for undergraduate students.
Project: 1. HBsAg Specific Immune Complexes of lgG Class in Feces of Patients with Hepatitis B
2.Fresh Water Fish Immune IgG Complexes Study.
Scientific Summary:
HBV is high occur rate in Chinese, and HbsAg is a good indicate for detect HBV infection. This study involved in developing new HBV complexes detection tool for clinic.
Fresh water fish is main protein resource for the people in China, especial in Southern China area. Study the immune system is so important both in basic biology and applied biology. Because the traditional concept is that fish has total different immune system with mammals.
Techniques Employed:
Serum collection from HBV patients (so we already got HBV vaccine before involved this project). ELISA.
COMPUTATIONAL Skills:
Utilization of Web-based gene prediction software and sequence alignment tools (e.g. GENSCAN, GrailEXP, HMMgene, MZEF, FirstEF, BLAST, CLUSTAL W)
Vector NTI Suite 9 alignment/assembly software
Sequin v3.85 sequence submission tool
ENSEMBL, Celera and UCSC Genome Browser utilization
Protein Structure Classification Websites (CATH, SCOP)
Microsoft Word, Excel, Powerpoint
PUBLICATIONS:
1. Keping Hu, Nan Xingsheng, André Bird, and Weidong Wang. Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex. Nature Genetics - 38, 962 – 964(2006)
2. Jifang Tao*, Keping Hu*, Qiang Chang*, Hao Wu, Nicholas E. Sherman, Keri Martinwich, Robert Klose, Carolyn Schanen, Rudolf Jaenisch#, Weidong Wang#, Yi Eve Sun#. Phosphorylation of MeCP2 at serine 80 regulates its chromatin association and neurological function. Proc. Natl Acad. Sci. USA. 106(12), 4882-4887, 2009 (*, # contributed equally to this work, this is a direct submission paper, a cover story was followed by Huda Zoghbi at the same issue of PNAS, title: The yin and yang of MeCP2 phosphorylation)
3. DongMei yang, KePing Hu, ChenYu Li, CaiHong Wu, and PeiAi Zhou. Neural electrophysiological effect of crude venom of Couns textile from the South China Sea Toxicon 2000 (38): 16071612.
4. KePing Hu, DongMei Yang, ZouJun Lin, YongCan Zhou, CaiHong Wu, PaiAi Zhou, Dominic Chan, and BingGen Ru. A new cysteine framework of conotoxin from Couns betulinus ( accepted by FEBS. Lett. but need more bioassay data).
5. Hu K, Lin F, Yang D, Zhang S, Zhou P, Wu C, Lin Z, Ru B, Chen J. The research proceeding of the biological activities of Conotoxins. Journal of Sanitary Toxtecology, 1997, 11(1): 35-37.
6. Peng Xuanxian, Wan Yange, Wang Sanying, Hu Keping. HBsAg specific immune complexes of lgG class in feces of patients with hepatitis B, Chinese J. Microbiol. & Immun. 1991(11):242245
10
Keping Hu, Ph.D. hukeping @gmail.com
7.Peng Xuanxian, Wu Lan, Hu Keping, Li Siguang. Binding properties between staphylococcal Protein A(SPA) and serum IgG of some animals species. Jiangxi Science. Science. 1992, Vol.10(4):246250.
8. Peng Xuanxian, Luo Hongqin, Chen Yanping, Zhou Yancan, and Hu Keping. Experimental study of protective action of shellfish mucus to burned. Jiangxi Science. 1992, Vol.10(2): 115-118.
INVITATION TALK ON SYMPOSIUM:
1. GTCbio presents. Topic: Role of Phosphorylation in Regulation of MeCP2 Function. September 18 & 19, 2006, Washington, DC.
2. 36TH the Society for Neurosciences (SfN) Conference. Topic: MeCP2 phosphorylation mapping and its function studies in seizure mouse and rat. “MeCP2 Function in Activity Dependent Plasticity, Brain Maturation, and Rett Syndrome”, in Oct 14-18, 2006, Atlanta.
3. 6th Annual Rett Syndrome Symposium. Topic: MeCP2 is regulated by phosphorylation and does not stably associate with SWI/SNF chromatin-remodeling complexes. (Authors: Keping Hu, Jifang Tao, Keri Martinowich, Hao Wu, Wenyu Zhu, Nickolas E. Sherman, Yi Sun, and Weidong Wang). June 27-29, 2005, Chicago.
POSTERS ON SYPMSOIUMS:
1. Keping Hu, Nicholas E. Sherman, Yi Sun, Weidong Wang, and Carolyn Schanen. MeCP2 Phosphorylation Mapping for Seized Mouse and Rat, 7th Annual RettSyndrome Symposium, June 26-28, Chicago.
2. Keping Hu and Weidong Wang. Purification and characterization of the Rett Syndrome protein, MeCP2. 5th Annual Rett Syndrome Symposium (p29), June28-30, 2004, Baltimore, MD.