Heng Zhu

Department Affiliation: Primary:  Pharmacology and Molecular Sciences;
High Throughput Biology Center (HIT Center)
Degree: Ph.D., Clemson University
Rank: Associate Professor
Telephone Number: 410-502-0878
Fax Number: 410-502-1872
E-mail address:  hzhu4@bs.jhmi.edu
Home Page:  http://www.jhu.edu/zhuheng/
School of Medicine Address:  Room 333, Edward D. Miller Research Building, 733 N. Broadway, Baltimore, MD 21205
 

Systems biology, construction of networks and pathways, transcription regulation, and pathogen-host interactions.

Fueled by the ever-growing DNA sequence information, the field of proteomics has become one of the most important disciplines to characterize protein function and activity and provide insights into structure of networks and pathways. The goal of my laboratory is to understand how proteins work together to achieve complex biological functions on the proteome level. To achieve this goal, we have developed two key technologies:  functional protein microarrays of high content and high density and a suite of biochemical assays for analyzing the properties of proteins. So far, we have fabricated proteome chips in budding yeast, herpesviruses, E. coli (K12), and humans. In parallel, we have developed various assays on the protein chips to characterize various protein binding properties, including protein-protein, protein-DNA, protein-lipid, and protein-drug interactions, and to identify downstream targets of protein kinases.

1. Construction of protein-DNA interaction networks in humans

To globally profile protein-DNA interactions in humans, we developed a protein microarray-based approach. First, a human protein microarray that contains the majority of annotated transcription factors and proteins in several other families was constructed. Second, we collected 400 and 60 predicted and known DNA motifs, respectively, labeled them with fluorescent dyes, and probed them individually to the protein microarrays. Third, using bioinformatic analysis, we not only characterized protein-DNA interactions for known transcription factors, but also unexpectedly found that hundreds of proteins not previously annotated as transcription factors also showed sequence-specific protein-DNA interactions.  These proteins represented a broad range of functional groups, including chromatin-associated proteins, transcriptional co-regulators, RNA-binding proteins, mitochondrial proteins, and protein kinases. Focusing on one such un-conventional DNA-binding protein Erk2, a well-studies MAP kinases, we confirmed its direct interaction with specific DNA sequences in cells, mapped the DNA-binding domain in Erk2, and demonstrated that the DNA-binding activity of Erk2 is solely responsible for transcription repression of the interferon-induced genes in vivo.  This study has profound impacts on the transcription field, because it suggests that there are probably more un-conventional DNA-binding proteins in humans that directly regulate transcription and therefore, greatly increasing the repertoire of transcription regulators. Part of this work was published in Cell.

2. Characterization of PTMs on lysine residues

Another major focus of our group is to dissect function of those lysine modification enzymes, including ubiquitin E3 ligases, SUMO E3 ligases, and histone acetyltransferases. Ubiquitylation is one of the most prevalent protein posttranslational modifications (PTMs) in eukaryotes; however, the underlining molecular mechanism still remains largely unknown.  To undertake this challenge, we developed in vitro ubiquitylation reactions with different E3 ligases on the yeast proteome chips and identified 86 candidate substrates for a HECT domain E3, Rsp5. Using in vitro and in vivo biochemical analyses, we were able to validate eight in vivo substrates of Rsp5.  By analyzing the effects of Ubp2, a deubiquitylating enzyme that associates with and counteracts Rsp5 activity, we found that the accumulation of the K63-linked poly-ubiquitin chains caused the processing of two new substrates. Drug sensitivity data also support the role of Rsp5 in positively regulating these substrates. This result is further supported by the observation that the cellular localization of a newly identified substrate Rnr2 is Rsp5-dependent. Part of this work was published in Molecular and Cellular Proteomics.

Histone acetyltransferases (HATs) and deacetylases (HDACs) conduct critical functions through nonhistone substrates in metazoans. However, nonhistone substrates have not been reported in S. cerevisiae. Using the yeast proteome microarray and an in vivo assay developed in the lab, we identified many nonhistone substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex and validated many novel substrates using in vitro and in vivo approaches. Among them, we identified the acetylation site (K514) of phosphoenolpyruvate carboxykinase (Pck1p) and showed that Esa1p (HAT) and Sir2p (HDAC) specifically catalyze the reversible acetylation and deacetylation at K514 in vivo.   Further, when K514 was mutated to arginine (K-to-R), the enzymatic activity of Pck1p was markedly decreased in vitro, and the cells lost the ability to grow on non-fermentable carbon sources. Consistent with the observation, K514 to glutamine (K-to-Q) mutation completely rescued the enzymatic activity and growth defect on esa1-531 background. More strikingly, the K514Q mutation resulted in longer life span under water starvation. Therefore, this study provides the first molecular evidence as how Sir2 plays a role in yeast longevity. Part of this work was published in Cell.

3. Pathogen-host interactions

It has been well documented that many RNA viruses encode functional RNA structures that interact with host proteins.  However, identifying proteins that bind specific RNA structure is a challenging task.  Therefore, we developed a new assay for RNA-protein interactions by probing the yeast proteome chips with a small RNA hairpin that contains a clamped adenine motif (CAM) encoded by a positive-stranded RNA virus, Brome Mosaic Virus (BMV). Among a dozen of hits, we focused on Pseudouridine Synthase 4 (Pus4) and the Actin Patch Protein 1 (App1) and found that they both modestly reduced BMV genomic RNA accumulation and translation, but dramatically prevented systemic spread in the infected plants. Pus4 prevented the encapsidation of BMV RNA3 in plants while App1 was inhibitory by a different mechanism. These results demonstrate the feasibility of using proteome arrays to identify specific RNA-binding proteins with potentially useful applications.

Herpesviruses are major human pathogens that cause life-long persistent infections with clinical manifestations that range from a mild cold sore, to lymphoma and angiogenic cancer.  Like other pathogens, they have a remarkable ability to usurp the host cell machinery for their propagation and spread. Our goal is to identify and characterize the important players to better understand the underlining mechanisms. To this end, we have generated an EBV protein array to systematically evaluate the targets of the EBV protein kinase, BGLF4.  The array experiments identified multiple proteins involved in EBV lytic DNA replication and virus packaging and assembly as previously unrecognized substrates for BGLF4.  These findings illustrate the broad role played by these protein kinases during lytic infection.  An unexpected discovery was the identification of EBNA1 as a substrate and binding partner of BGLF4.  EBNA1 is essential for replication and maintenance of the episomal EBV genome during latency.  In functional assays, we showed that BGLF4 was recruited by EBNA1 to the EBV latency origin of replication, oriP, and inhibited oriP-dependent DNA replication/maintenance.  Experiments using a kinase null BGLF4 mutant revealed that the kinase activity of BGLF4 was required for this inhibition.  Recent therapeutics have focused on developing nucleoside analogs and lytic inhibitors (e.g. ganciclovir and maribavir) to limit herpesvirus replication but these agents do not affect latently infected cells.  Our data raise the possibility that EBV genomes can be eliminated from latently infected cells by BGLF4 induction, thus providing a novel therapeutic strategy for EBV associated malignancies.

Representative Publications:

• Song Q, Liu G, Hu S, Zhang Y, Tao Y, Han Y, Zeng H, Huang W, Li F, Chen P, Zhu J, Hu C, Zhang S, Li Y, Zhu H*, Wu L. Novel Autoimmune Hepatitis-Specific Autoantigens Identified Using Protein Microarray Technology. J Proteome Res 2010; 9(1):30-9. Pub Med Reference

• Hu S, Xie Z, Onishi A, Yu X, Jiang L, Lin J, Rho H-S, Woodard C, Wang H, Jeong J-S, Long S, He X, Wade H, Blackshaw S, Qian J, Zhu H*. Profiling the Human Protein-DNA Interactome Reveals MAPK1 as a Transcriptional Repressor of Interferon Signaling. Cell 2009; Oct 30;139(3):610-22. Pub Med Reference

• Kung L, Tao, S-C, Qian, J, Smith M, Snyder M, Zhu H*. Global analysis of the glycoproteome in S. cerevisiae reveals new roles for protein glycosylation. Mol Syst Biol 2009; 5:308. Pub Med Reference

• Chen J, Sullivan S, Anderson T, Tan SC, Alex, PJ, Brant S, Taylor MV, Burek L, Cuffari C, Wang H, Li R, Datta LW, Winslow R, Zhu H, Li X.  Identification of novel serlogical biomarkers for inflammatory bowel disease using a protein chip of whole E. coli proteome. Mol Cell Proteomics 2009; 8(8): 1765-76 Pub Med Reference

•  Zhu J, Liao G, Shan L, Zhang J, Chen M-R, Hayward GS, Hayward SD, Desai P, Zhu H. Protein array identification of substrates of the Epstein-Barr Virus protein kinase BGLF4.J Virol 2009; 83:5219-31. Epub 2009 Feb 25. Pub Med Reference

• Lin Y-y, Lu J-y, Zhang J, Walter W, Dang W, Wan J, Tao SC, Qian J, Zhao Y, Boeke JD, Berger SL, Zhu H. Protein acetylation microarray reveals NuA4 controls key metabolic target regulating gluconeogenesis.Cell 2009; 136:1073-1084. Pub Med Reference

• Tao SC, Li Y, Zhou J, Qian J, Schnaar RL, Zhang Y, Goldstein IJ, Zhu H, Schneck JP. Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers.Glycobiology 2008; 18:761-9. Epub 2008 Jul 14. Pub Med Reference

• Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y. Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli. Mol Cell Proteomics 2009; 8:215-25. Epub 2008 Aug 23.Pub Med Reference

• Liu X, Yu X, Zack DJ, Zhu H, Qian J. TiGER: a database for tissue-specific gene expression and regulation.BMC Bioinformatics 2008; 9:271. Pub Med Reference

• Chen C-S, Korobkova E, Chen H, Zhu J, Jian X, Tao S-C, He H, Zhu H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.Nature Methods 2008; 5:69-74. Epub 2007 Dec 16. Pub Med Reference

• Lu, J.-Y., Lin, Y.-Y., Tao, S.-C., Zhu, J., Pickart, C.M., Qian, J., and Zhu H*.  Functional dissection of a HECT ubiquitin E3 ligase, Mol. Cell Proteomics 2008, 7:35-45. Pub Med Reference

• Zhu, J., Gopinath, K., Murali, A., Yi, G., Hayward, S.D., Zhu, H., Kao, C. RNA binding proteins that inhibit RNA virus infection, Proc. Natl. Acad. Sci. USA 104:3129-3134, 2007. Pub Med Reference
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• Tao, S.-C. and Zhu, H. Protein chip fabrication by capture of nascent polypeptides, Nature Biotech. 24:1253-1254, 2006. Pub Med Reference

Other graduate programs in which Dr. Zhu participates:

BCMB Graduate Program