Keping Hu, Ph.D.
hukeping @gmail.com
Keping Hu, Ph.D.
Email: hukeping@gmail.com
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-ofthe 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
1
Keping Hu, Ph.D.
hukeping @gmail.com
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
2
Keping Hu, Ph.D.
hukeping @gmail.com
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
3
Keping Hu, Ph.D.
hukeping @gmail.com
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 Toxinducts of Conus,
2. Biosynthesis and Chemical Purification of QS, a
Carboxylase specific substrate.
4
Keping Hu, Ph.D.
hukeping @gmail.com
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 Cai-Hong 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.
5
Keping Hu, Ph.D.
hukeping @gmail.com
VISITING SCHOOLAR , 10/93 to 5/94
National Laboratory of Protein Engineering, Peking University,
Beijing, China
PI: Bing-Gen 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-molecularweight, 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:
6
Keping Hu, Ph.D.
hukeping @gmail.com
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 brahmaassociated 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), 48824887, 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. Dong-Mei yang, Ke-Ping Hu, Chen-Yu Li, Cai-Hong Wu, and
Pei-Ai Zhou. Neural electrophysiological effect of crude venom
of Couns textile from the South China Sea Toxicon 2000 (38):
1607-1612.
4. Ke-Ping Hu, Dong-Mei Yang, Zou-Jun Lin, Yong-Can Zhou,
Cai-Hong Wu, Pai-Ai Zhou, Dominic Chan, and Bing-Gen 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):242-245
7
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):246-250.
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. 5 th
Annual Rett Syndrome Symposium (p29), June28-30, 2004,
Baltimore, MD.
3 Keping Hu and Weidong Wang. Immunoprecipitation of
MeCP2 suggested that significant amount of endogenous
MeCP2 do not associate with histone deacetylase. 4th Annual
Rett Syndrome Symposium (p36). June 23-25, 2003, Baltimore,
MD
4. Keping Hu, Dongmei Yang, Caihing Wu, Peiai Zhou.
Modulation of neuronal Na+ channels activities by conotoxin. 5 th
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Keping Hu, Ph.D.
hukeping @gmail.com
IBRO World Congress of Neuroscience, Jerusalem, Israel, July
11-15, 1999, p148.
REFERENCES
N. Carolyn Schanen, M.D., Ph.D.
Head of Human Genetics Research Laboratory
Nemours Biomedical Research
Rockland Center I Room 241
1701 Rockland Road
Wilmington DE 19803
(302) 651-6702
(302) 651-6767 FAX
schanen@medsci.udel.edu
Dr. Yi Sun, Associate Professor
David Geffen School of Medicine at UCLA
Neuroscience Research Building Suite 355
635 Charles E. Young Dr. South
Los Angeles, CA90095
Office: 310-825-9506
Lab: 310-267-2197
Email: ysun@mednet.ucla.edu
Dr. Weidong Wang, Section Chief
Lab of Genetics, National Institute on Aging/NIH
333 Cassell Dr. Suite 3000
Baltimore, MD 21224
Tel: 410-558-8334
Email: wangw@grc.nia.nih.gov
Dr. Bruce D. Korant
Research Fellow
Dept of Biochemistry
AI duPont Hospital for Children
Nemours Foundation
Rockland Bldg One rm 229
1600 Rockland Road
Wilmington Del USA19803
tel
302 651 6723
fax
302 651 6767
email bkorant@nemours.org
Prof. Adrian Bird, Ph.D. Deputy Chair, Wellcome Trust,
Director of the Wellcome Trust for Cell Biology,
Institute of Cell and Molecular Biology
9
Keping Hu, Ph.D.
hukeping @gmail.com
University of Edinburgh
Michael Swann Building
King's Buildings
Mayfield Road
Edinburgh EH9 3JR
Scotland U.K.
A.Bird@ed.ac.uk
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