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Molecular & Cellular Proteomics 6:S65-S66, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstracts

Session 7

Sperm Chromatin Proteomics Identifies Evolutionarily Conserved Fertility Factors

D. Chu¹, H. Liu^2,3, J. Yates², B. Meyer^4,5

¹Department of Biology, San Francisco State University, San Francisco, CA; ²Department of Cell Biology, Scripps Research Institute, La Jolla, CA; ³Agilent Technologies, Inc., Wilmington, DE; ⁴Howard Hughes Medical Institute, Berkeley, CA; ⁵Department of Molecular and Cell Biology, University of California, Berkeley, CA

Human male infertility and in vitro fertilization failure can arise from defects in sperm chromatin composition and structure. To understand the underlying molecular causes of male infertility, we combined proteomics, cytology, and functional analysis in Caenorhabditis elegans to identify conserved spermatogenesis-enriched chromatin proteins important for fertility. Our strategy involved purification of chromatin from C. elegans spermatogenic or oogenic germ cells, identification of chromatin proteins through Multidimensional Protein Identification Technology, subtraction of common proteins, and prioritization of spermatogenesis-enriched proteins for functional analysis based on abundance. This approach reduced 1099 spermatogenesis-enriched chromatin proteins, the most extensive catalog of such proteins available, to 132 proteins for analysis. Functional analysis in C. elegans revealed conserved spermatogenesis-specific proteins are vital for DNA compaction, chromosome segregation, and fertility. Proteins with diverse functions in other cell types showed enrichment on sperm chromatin, revealing unexpected roles for general factors in spermatogenesis. Of 25 candidates with genetically disrupted mouse homologs, 11 cause infertility in male mice, validating our strategy to identify conserved fertility factors. Our list provides significant opportunity to identify causes of male infertility and targets for male contraceptives.

7.2

Walking in the Proteomic Footprints of Viral Infection

I. M. Cristea1¹, M. P. Rout², B. T. Chait¹

¹Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, ²Laboratory of Cellular and Structural Biology, Rockefeller University, New York, NY

Protein interactions, of stable or transient nature, underline the majority of cellular processes. Through such interactions, viruses succeed in sabotaging complex organisms and turning their cellular machineries to their own use. Although many virus-host interactions have been studied, our knowledge of the interactions between viral and host proteins remains quite limited. We have recently developed a genomic-proteomic approach to study the dynamic localizations and interactions of viral proteins during the course of viral infections. This presentation will report on technical aspects of our strategy and results from our studies of Sindbis virus and human cytomegalovirus (HCMV). Our findings led to hypotheses as to how viruses manipulate host cellular processes. These studies demonstrate the importance of proteomic approaches in helping us gain a better understanding of the molecular details of viral infections, and just as importantly, the biology of the cell.

Our approach has the following steps: (1) Creation of replication-competent mutant viruses, genomically tagged with green fluorescent protein, protein A, or FLAG. (2) Natural infection of mammalian cells followed by fluorescence microscopy to visualize tagged viral proteins at various stages of infection. (3) Immediate Freezing of the cells followed by lysis in their cryogenic state to help maintain protein complexes close to their original condition in the cell. (4) Rapid immunoisolations (5–60 min) using magnetic beads coated with antibodies against the tag. (5) Identification of the isolated proteins by mass spectrometry. (6) Confirmation of the observed interactions by co-localization and reciprocal immunoisolation of the host proteins. (7) Further studies are conducted for the functional characterization of the interactions.

Using this approach, our studies of the Sindbis virus, an Alphavirus genus member that in humans causes arthritis, provided details of temporally-regulated viral-host interactions during the course of infection. One resulting hypotheses is that Sindbis utilizes G3BP, a putative nuclear transport factor, to capture host RNAs undergoing nuclear export, for subsequent sequestration in structures involved in translational shut off. Additional studies of the Alphavirus Ross River and Flavivirus Yellow Fever viruses indicated that G3BP may be generally important during Alphavirus infection.

We have extended our studies to other viral systems, including HCV, BVDV, and HIV. Most recently, we have initiated a comprehensive study of the HCMV interactome. HCMV establishes a latent infection in 80% of adults by the age of 40. Congenital infection with HCMV is the leading viral cause of birth defects in U.S.A. To study the HCMV interacting network, we have constructed a library of 155 tagged HCMV viruses. Results from our studies of the early infection environment will be presented. The functional significance of the observed interactions will be discussed, including our finding that HCMV ensures cell growth and ribosome biogenesis by blocking normal cellular responses to stress.

7.3

Exploiting Viruses to Identify Critical Interactions and Therapeutic Targets in the Cellular Networks That Regulate Growth and Survival

C. Soria¹, A. Miller², K. Espantman¹, J. Smyth², R. Chalkley², A. Burlingame², C. O'Shea¹

¹Salk Institute for Biological Studies, MCBL, La Jolla, CA; ²Department of Pharmaceutical Chemistry, University of California, San Francisco, CA

Protein-protein interactions provide a framework for understanding biology and disease at an integrated and mechanistic level. A major challenge is to define the critical cellular hubs and interactions that are subverted to elicit growth deregulation, a phenotype that is at the heart of many diseases, including cancer.

Unfortunately, human tumors acquire a myriad of molecular lesions, which makes it difficult to define the critical therapeutic targets. In contrast, the small DNA tumor virus, adenovirus, encodes 16 early growth deregulatory proteins that have been selected over millions of years to target the critical cellular hubs that control human cell growth and survival. It is the very same cellular networks that are disrupted by mutations in cancer. As such, there is a striking overlap between the key cellular targets of tumor mutations and DNA viral proteins. Indeed, many of the critical growth regulatory targets, for example p53, E2F and PI-3 kinase, were initially identified in studies with DNA viral proteins. Proteomics has progressed a long way in the interim. Tandem Affinity Purification (TAP) combined with Mass Spectrometry is a powerful approach to defined the cellular targets of early adenoviral growth deregulatory proteins, which have yet to be systematically identified.

We are exploiting adenovirus as a powerful paradigm for an integrative systems understanding of a natural, dynamic and pleiotropic growth deregulation program in normal human cells. In collaboration with Dr. Alma Burlingame's lab at UCSF, we are comprehensively identifying the cellular interactions targeted by early adenoviral proteins both in uninfected and infected cells. This is providing new insights into key growth regulatory pathways and how they could be modulated for cancer therapy, such as, PI-3 kinase/mTOR and p53.

In adenovirus infection, E1B-55K mimics mdm2 in binding and degrading p53. On this basis an adenovirus mutant lacking E1B-55K (ONYX-015) was proposed and tested as a p53-selective cancer therapy. However, we found that p53 plays a minor role in determining the tumor selectivity of this virus. This is because, although the absence of E1B-55K results in very high levels of p53 in infected primary cells, p53 activity is nonetheless suppressed even upon irradiation. Recently, to determine the mechanism whereby p53 is inactivated, we screened for p53 activation in primary cells infected with adenoviruses that have compound mutations in E1B-55K and other early viral genes. These studies have revealed that there are additional early viral genes which dominantly suppress p53 activity in the absence of p53 degradation. Using TAP tags and mass spectrometry, we have identified novel cellular proteins that interact differentially with active versus inactive p53 in E1B-55K mutant virus infected cells and upon adriamycin treatment. The results of these studies have important implications for understanding p53 regulation and how it could be modulated for tumor therapy.

7.4

High Throughput Characterization of Amplified Nucleic Acids by ESI-TOF Mass Spectrometry: Applications in Pathogen Detection and Characterization

S. A. Hofstadler

Ibis Biosciences, Carlsbad, CA

High throughput ESI-TOF analysis of amplicons represents a novel and universal strategy for the detection and characterization of microorganisms associated with emerging infectious diseases. The process uses mass spectrometry, signal processing, and base composition analysis of PCR amplification products from biologically conserved regions of microbial genomes to simultaneously identify the organisms present in a sample without the need for culture. The sample can be derived from environmental samples, clinical specimens, or other sources. Core to this approach are broad range PCR primers that target broadly conserved regions of microbial genomes that flank variable regions. Using high-performance electrospray ionization mass spectrometry (ESI-MS), the base composition (i.e., the number of As, Gs, Cs, and Ts) of each amplicon is unambiguously determined.

Bacterial Example: We have examined cultures and direct throat swabs obtained from individuals suspected to be suffering from Group A Streptococcus(GAS) infections. Samples were first analyzed using a panel of survey primers that readily identified the infectious agent as Streptococcus pyogenes, clearly distinguishable from all other organisms, including other Streptococciand Staphylococci. Subsequent analysis with Streptococcus-specific primers rapidly yielded emm-types for each sample which were later corroborated with conventional MLST analyses. This study demonstrated that this approach can be used to detect and identify infectious agents directly from a throat swabs. In the present configuration, hundreds of samples can be analyzed within 12 hours allowing near real-time evaluation of patient samples and will make possible more rapid and appropriate treatment of patients in an ongoing epidemic. The use of "drill down" primers allows closely related strain variants to be distinguished and accurately identified. This is of particular importance when tracking the spread of particularly virulent strains of disease-causing organisms.

Viral Example: The base compositions of amplicons from six influenza genes were used to provide sub-species identification and infer H and N subtypes. The method detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, avian H5N1 isolates. Further, PCR/ESI-MS enabled correct identification and mapping of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) to previously established clades. Thus, PCR followed by rapid ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations, and monitor viral evolution. This method promises to become an integral component of high-throughput influenza surveillance.

This strategy distinguishes this approach from other detection/identification strategies in that it requires little or no prior knowledge about an organism in order to identify it in a sample. The approach requires that high-performance mass measurements be made on PCR products in the 80–140 bp size range in a high-throughput, robust modality. The base compositions from multiple primer pairs are used to "triangulate" the identity of the organisms present in the sample. Use of species-specific primers allows rapid strain-typing of the organism. The concept is equally applicable to bacteria and viruses and has recently been applied to detection of variations in human mtDNA associated with mitochondrial diseases.

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