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Using Proteomics to Understand Avian Systems Biology and Infectious Disease¹

^* Department of Animal Science, North Carolina State University, Raleigh 27695-7621

² Corresponding author: hc_liu{at}ncsu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
The proteome is defined as the protein complement to the genome. Proteomics is the study of the proteome. Several techniques are frequently used in proteomics; these include 2-hybrid systems, 2-dimensional gel electrophoresis, and mass spectrometry. Systems biology is a scientific approach that takes into account the complex relationships among and between genes and proteins and determines how all of these interactions come together to form a functional organism. Proteomic tools can simultaneously probe the properties of numerous proteins and thus are a great aid to the emerging field of systems biology, in which the functional interactions of numerous proteins are studied instead of studying individual proteins as isolated entities. In the field of avian biology, proteomics has been used to study everything from the development and function of organs and systems to the interactions of infectious agents and the altered states that they induce in their hosts.

Key Words: proteomics • systems biology • infectious disease

	INTRODUCTION

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
The word proteome was originally coined to describe the protein complement to the genome (Wasinger et al., 1995). Proteomics is the study of the proteins that make up the proteome. Three of the most popular proteomic tools are the yeast 2-hybrid system, 2-dimensional gel electrophoresis, and mass spectrometry. Another useful proteomics tool has been the development of comprehensive protein databases that are crucial for the analyses of data that are generated by these proteomic tools.

The yeast 2-hybrid system was originally developed as a genetic means for identifying and characterizing the interaction between 2 proteins. The yeast 2-hybrid system also provides a powerful and complementary approach to classical biochemical methods, such as coimmunoprecipitation and electrophoretic mobility shift analysis, for characterizing protein-protein interactions (Fields and Song, 1989). Today the yeast 2-hybrid system has become a popular method for global screening of protein interactions. The simplified concept of the yeast 2-hybrid system is based upon the utilization of the yeast protein GAL4 transcription factor, which functions to regulate the expression of galactolytic enzymes and consists of 2 distinct and independent functional domains. The first domain binds to a specific DNA sequence near the promoter from which the galactolytic enzymes are expressed, whereas the second domain functions to activate transcription by physically interacting with RNA polymerase. In the yeast 2-hybrid system, the "bait" protein is fused to the DNA-binding domain and the "prey" proteins are fused to the activation domain. If the bait and a prey interact, the 2 domains are brought together and reconstitute a functional GAL4 molecule, which can then activate expression of the reporter gene, which is often ß-galactosidase (reviewed by Miller and Stagliar, 2004). Yeast cells then grown in the presence of the chromogenic substrate X-gal will turn blue due to the hydrolysis of the X-gal by the newly expressed ß-galactosidase. Today, there are many variations of the yeast 2-hybrid system in use.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has become a popular and quite powerful way to characterize whole protein extracts of a particular sample. In 2D-PAGE, proteins are first applied to the end of a rod or strip of a polyacrylamide gel containing a pH gradient. An electric current is then applied to the gel and proteins are then separated in the first dimension by their charge, which is dependent on their isoelectric points. As a given protein migrates through the gel toward an electrode, the protein will enter a region of the pH gradient that equals its isoelectric point, and the protein will then assume a net charge of zero and will cease to migrate any further along the gel. Proteins in the second dimension are separated according to their mass. Separation is achieved by placing the gel rod containing the proteins separated in the first dimension onto a second polyacrylamide gel containing the anionic detergent sodium dodecyl sulfate. In the presence of the detergent, all of the proteins assume an equal charge to mass ratio and are then electrophoretically separated as proteins of higher masses migrate toward the anode more slowly through the gel than do proteins of lower masses. The gel is then stained so that the proteins can be visualized, and the stained gel reveals a map on which the position of each protein is determined by coordinates consisting of pH on the first axis and mass on the second axis (reviewed by Wittmann-Liebold et al., 2006). Two-dimensional gel electrophoresis is often used in conjunction with mass spectrometry to identify specific proteins of interest.

Mass spectrometry was originally developed in the early 1900s as a tool to study characteristics, such as mass and charge, of compounds. It is most often used to identify an unknown compound based on these characteristics. In mass spectrometry a sample is exposed to an ionizing source, such as a photon beam, which ionizes the sample. These ions are carried into a mass analyzer and are separated based on their mass-to-charge ratio. This separation leads to the production of a mass spectrum (the mass to charge ratios of all of the ions that compose the sample), which can then be used to identify the sample. Mass spectrometry is a vital component of proteomic analysis because it allows for the identification of proteins. High performance liquid chromatography is often used prior to mass spectrometry analyses, such as electrospray ionization mass spectrometry (ESI-MS), to separate peptides and proteins (reviewed by Listgarten and Emili, 2005). There are 2 phases in HPLC, a mobile (liquid) phase and a stationary phase. The liquid phase (contains the peptides) is forcibly passed through a column (stationary phase) containing small uniform particles. Separation is controlled by the interaction of the particles with the solutes present in the sample, and the peptides are separated based on their chemical properties. Once peptide separation is achieved, mass spectrometry is used to identify the peptides. Liquid chromatography is also often used in conjunction with tandem mass spectrometry (LC-MS/MS). In tandem mass spectrometry there is an initial scan of all of the ions in the sample followed by another mass spectrometry scan in which only certain ions are analyzed and used in determining the peptide sequence (Listgarten and Emili, 2005).

In proteomics one of the most frequently used mass spectrometry methods is matrix-assisted laser desorption/ionization (MALDI) coupled with time of flight (TOF). This mass spectrometry approach is often used in conjunction with 2D-PAGE. Interesting proteins identified by 2D-PAGE are excised from the gel and trypsin digested to create small peptide fragments. In MALDI the protein sample is dissolved on a solid matrix, and a laser light is directed onto the matrix, which absorbs the energy and vaporizes so the matrix and the sample are now ionized and in a gas phase (reviewed by Zaluzec et al., 1995). When used with TOF, the ionized sample is then passed into a TOF analyzer where smaller ions will move faster and reach the detector first. The mass spectrum produced can then be used to identify the peptides. These peptides are then searched against protein databases to identify the parent protein from which they originally came.

Another mass spectrometry technique that is often used in proteomics is ESI-MS. In ESI-MS a liquid sample is sprayed from a capillary in a strong electric field, which creates a spray of highly charged droplets (reviewed by Smith et al., 2006). These charged droplets are attracted toward the mass analyzer. As they move toward the mass analyzer, gas, heat, or both is applied to the droplets, causing them to decrease in diameter. This decreased diameter brings the ions closer together creating repulsion forces that exceed the surface tension forces, which causes ions to leave the droplet. The ions now enter the mass analyzer, and their mass to charges ratios can be determined.

	BENEFITS AND LIMITATIONS OF PROTEOMICS

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There are many benefits to the use of proteomics in the study of systems biology. For example, many proteomic techniques are high-throughput methods and can give extensive information on global protein expression. Often expression at the RNA level does not reflect expression at the protein level, so the use of proteomics can give a better idea of protein expression. Proteomic techniques such as 2D-PAGE and mass spectrometry can give rather detailed characterizations of proteins, giving not only their molecular weight but also revealing their isoelectric points and showing if the proteins are acidic, basic, or neutral. The techniques can also reveal posttranslational modifications, such as acetylation and phosphorlyation. By looking at the global protein expression of a particular tissue or cell type, new functions of proteins can be suggested, and more information about previously identified pathways or even new pathways can be discovered.

Though proteomics can give much information about protein expression, there are some limitations. For example, one of the most limiting factors in the use of proteomics is the lack of comprehensive databases (Han et al., 2005; Kuo et al., 2005; Lam et al., 2006). Characterizations of peptide sequences determined from mass spectrometry spectra are only useful if good protein databases exist for comparison. Another limitation in regard to mass spectrometry is the inability to identify a protein due to the small amount of starting material (Lam et al., 2006). Sometimes with mass spectrometry, ambiguous results make it impossible to definitively identify a protein (Han et al., 2005). Another large limitation found in proteomics is that the proteins identified depend on how the sample was solubilized. Different solubilization procedures could lead to different proteome maps of the same sample (Stagsted et al., 2004). So depending upon how protein solubilization was preformed, critical proteins could be lost. In regard to 2D-PAGE, often there is a lack of comparative reproducibility between gels due to operator and reagent variation. Despite these limitations proteomics has and will continue to be a good source of data for systems biology.

	SYSTEMS BIOLOGY

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
Classical molecular biological studies often look at an individual gene or protein as an isolated entity rather than as part of a complex system. Systems biology is a scientific approach that takes into account the complex relationships among and between genes and proteins and determines how all of these interactions come together to form a functional organism (reviewed by Aggarwal and Lee, 2003). Proteomics contributes to the study of systems biology by revealing protein-protein interactions as well as protein expression levels. For example, yeast 2-hybrid studies can reveal novel interactions and can therefore suggest pathways involved in cellular function or in a pathogenic infection. The use of 2-dimensional electrophoresis can create proteome maps of tissues, cells, etc. and can also provide the relative abundance of proteins present in a sample. The 2-dimensional electrophoresis system is often used in complement with mass spectrometry. Mass spectrometry can be used to determine masses as well as the sequences of peptides from a given protein. Proteomic techniques can be used to identify possible pathways that are involved in normal cellular function and in pathogen altered functions and thus can serve as one of the initial steps in the study of systems biology.

	CHICKEN AS A MODEL ORGANISM

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
Chicken embryos have long served as a model for development (Mann, 1921; Drake et al., 2005). The embryo is easily accessible, readily obtainable, and can be manipulated in vivo (in ovo) more easily than can mammalian embryos (Brown et al., 2003). A hen will typically lay many eggs a year, thus producing many more offspring than a typical mammal. In addition, chicken embryonic development from fertilization to hatching takes only 21 d, a developmental span that is of much shorter duration than that of most mammals, allowing for faster accumulation and interpretation of data.

	PROTEOMICS AND THE EGG

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
The egg is a complex structure that protects and nourishes the developing avian embryo. Very little work has been done to study the protein content of eggs because the abundance of a few major proteins makes it difficult; for example, ovalbumin composes more than 50% of the egg white (Guérin-Dubiard et al., 2006). To further characterize the minor proteins present in egg white, Guérin-Dubiard and colleagues used 2D-PAGE with a wide pH gradient (pH 3 to 10). These gels along with peptide mass fingerprinting (PMF) MALDI-TOF identified 16 minor egg white proteins, including 2 of which had not been found in egg white previously. These proteins included chaperones, protease inhibitors, a vitamin receptor, and antimicrobial proteins. One of the most interesting is Tenp, which had not previously been identified in egg white. This protein is thought to have an antimicrobial function; it binds lipopolysaccharides of the outer envelope of gram-negative bacteria. Proteomic analysis of the egg white revealed there are many minor proteins present that exhibit a wide variety of functions, many of which could be vital during chick development.

Egg production is a large economic part of the poultry industry worldwide. Understanding the processes and pathways that control egg production could greatly benefit this industry. In the chicken, hormones secreted by the hypothalamus control reproduction and therefore control egg production (Kuo et al., 2005). In their work, Kuo et al. used a proteomics approach to study and compare hypothalamic proteins in high-egg-producing birds vs. low-egg-producing birds in an attempt to discover new markers for selection of high egg production. Hypothalamic proteins from 2 strains of birds (originally from the same population) that differ in egg production rates were analyzed by 2D-PAGE and by HPLC and nano-ESI-MS. After comparing protein expression between the strains prior to and during egg production, 8 protein spots with differential expression were identified. Six of these spots were able to be identified by LC-MS/MS. One of the proteins in these spots was identified as HNRPH3, which is a member of a group of nuclear ribonucleoproteins that regulate gene expression at the mRNA level by transport, stability, and editing of mRNA (Kuo et al., 2005). The HNRPH3 was found to have a higher expression level in the hypothalamus of high egg production birds than low-egg-producing birds. It was thought perhaps HNRPH3 contributes to higher stability and expression of gonadal hormone mRNA in the hypothalamus, which, in turn, increases egg production. Using proteomics this study was able to identify a potential marker for high egg production in poultry.

	PROTEOME OF GERM CELLS

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
A proteome map of chicken gonadal primordial germ cells (gPGC) was determined to better understand germ cell development (Han et al., 2005). Two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry and LC-MS/MS of proteins isolated from in vitro cultured gPGC identified 61 proteins expressed in these cells. Most of these gPGC proteins that were identified had been previously characterized and thus allowed for the creation of a proteome map that revealed some idea about the various pathways that are involved in germ cell development (Han et al., 2005).

	PROTEOMICS AND FACIAL DEVELOPMENT

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
The chick embryo was recently used for proteomic profiling of facial development (Mangum et al., 2005). During vertebrate embryonic development the face develops from the first branchial arch. This is a very small structure from which only a small amount of tissue can be collected. Mangum and colleagues used the chick to study facial development because of the relative ease to obtain and maintain large numbers of embryos. Two-dimensional PAGE and MALDI-TOF mass spectrometry were used to identify 21 proteins (mainly chaperones involved in cell structure or associated with vascularization) that exhibited differential expression between days E3 and E5 of chick development. However, overall very little change in protein expression was seen, suggesting that during this time phase in development there is more growth than differentiation in the first brachial arch (Mangum et al., 2005).

	PROTEOMICS AND THE EYE

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
As with many other aspects of development, the chick is a common model organism used for eye development. The retina plays a role in regulating eye growth during development in addition to its role in vision. Understanding protein expression during eye development could better enable the understanding and treatment of degenerative eye disorders and blindness. Developing proteomic profiles of retinal protein expression during various stages of development will better assist the study of the vertebrate eye. Two-dimensional PAGE, MALDI-TOF, and peptide mass fingerprinting were utilized to profile retinal proteins at d 3, 10, and 20 posthatch (Lam et al., 2006). This study was successfully able to identify 155 proteins expressed during eye/retinal development. Based on their functions these proteins were classified into 10 gene ontology categories. In gene ontology, genes are classified into categories based on their function (http://www.geneontology.org/index.shtml). Most of the proteins were found in the catalytic activity, binding, or transporter activity groups. This suggests that metabolic functions as well as signalling and transduction functions are very important during vertebrate eye development.

Another study used the chicken as a model to study the lens crystallins proteome (Wilmarth et al., 2004). It is known that the lens in vertebrates is mainly made up of crystallins. Proteomic tools were used to study the different isoforms of the crystallin proteins in the chicken eye. After 2D-PAGE and MS/MS analysis it was found that there are 2 isoforms of crystallin ( A and B), as well as a phosphorylated isoform of B and 4 isoforms of ß crystallin, 3 phosphorylated isoforms (ß A1, ß A2, ß B1), and 1 isoform of ß B3 (Wilmarth et al., 2004). The DNA sequence analysis revealed that these isoforms arise from alternative splicing and posttranslational modifications such as acetylation and phosphorylation.

	PROTEOMICS AND MUSCLE

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
Proteome changes that occur during skeletal muscle development have been characterized using 2D-PAGE and MALDI-TOF (Doherty et al., 2004). Understanding what proteins and pathways are involved in avian muscle development will not only be a tremendous benefit to the poultry industry but will also extend our understanding of vertebrate muscle development. In the studies of Doherty et al. (2004), proteome profiles of the layer pectoralis (breast) muscle at 5 different developmental time points posthatch (1, 5, 9, 13, and 27 d) were determined. Two-dimensional PAGE gels demonstrated similar protein expression patterns among earlier time points and then a shift occurring around 5 d posthatch followed by a similar pattern among later stages. Earlier developmental time points exhibited a larger variety of protein expression, whereas later stages appeared to have more specialized protein expression. During the later stages the highly expressed proteins included several glycolytic enzymes that participate in energy metabolism. This higher degree of specialization could be due to the large muscle mass present in later stages and that have a higher energy requirement.

Another study used 2D-PAGE and MALDI-TOF mass spectrometry in concordance with an immunologic approach to look at oxidized proteins in the chicken pectoralis muscle (Stagsted et al., 2004). This knowledge is useful because understanding and controlling protein oxidation in muscle tissue could lead to better meat quality. In this work, antibodies specific for carbonyl groups and 3-nitrotyrosines (specific for oxidized proteins) were used to detect oxidized proteins in both 1D- and 2D-PAGE. Identified oxidized proteins were then further analyzed by MALDI-TOF, which revealed that the oxidized proteins were -enolase, actin, heat shock protein 70, and creatine kinase. Alphaenolase was abundantly expressed in the pectoralis and thigh muscles and was also extensively oxidized in both. This study identified the oxidation of enolase as a possible marker of the antioxidative status of muscle.

	PROTEOMICS AND THE AVIAN IMMUNE SYSTEM

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
Avian species possess a variety of genetic components that are involved in susceptibility or resistance to certain infectious agents. For example, chickens with the B21 haplotype are resistant to Marek’s disease, and chickens with the B19 haplotype are susceptible. Several proteomic studies have been undertaken to determine what peptides are presented by these 2 haplotypes and to identify the differences in peptide-receptor binding motifs between the haplotypes (Haeri et al., 2005; Cumberbatch et al., 2006). The HPLC fractionation and MALDI/MS identified 4 peptides associated with the MHCII from the B21 haplotype and 1 peptide from the B19 haplotype (Haeri et al., 2005). Proteins identified from the B21 haplotype were from the cytosol, membrane, and mitochondria, whereas the peptide identified from the B19 haplotype was cytosolic. However, too few proteins were identified to determine an antigen-binding motif for these MHC molecules. Another study looking at peptides presented by the B19 haplotype identified 30 peptides that were presented by this MHC haplotype (Cumberbatch et al., 2006). Peptides present in the plasma membrane, cytosol and endosome, were identified as well as peptides from the envelope glycoprotein of chicken syncytial virus. The analysis of these peptides was used to identify a putative core-binding motif for the B19 MHCII molecule. By using proteomic approaches to identify peptides presented by different MHCII haplotypes and to determine their peptide binding motifs, differences in the antigenic presentation can be discovered, perhaps leading to the explanation for why some haplotypes are more susceptible or resistant than others.

To create a functional model of the bursa of Fabricius, a study that used differential detergent fractionation-multidimensional protein identification technology was used to identify almost 6,000 proteins expressed in the bursa (McCarthy et al., 2006). Differential detergent fractionation uses a series of detergents to separate cytosolic, membrane, and nuclear proteins. By superimposing these proteins over previously characterized mammalian pathways, putative pathways in the chicken were determined. These pathways included those involved in programmed cell death (apoptosis) and cell proliferation and differentiation.

Another area where proteomics has been useful is in the study of antimicrobial substances. A study of antimicrobial proteins present in the chicken reproductive system discovered 2 proteins with antimicrobial activity in the ovary and oviduct (Silphaduang et al., 2006). Isolation and partial sequencing of 2 peptides by tandem nanoelectrospray mass spectrometry revealed that histones H1 and H2B were the antimicrobial proteins. In different study of antimicrobial proteins from Martín-Platero et al. (2006), the uropygial gland of birds is a preen gland that produces a hydrophobic fatty acid secretion. A bacterium, Enterococcus faecalis, has been isolated from the uropygial gland and has been shown to have antimicrobial properties. Two antimicrobial peptides from E. faecalis were isolated and subjected to MALDI/TOF analysis. The analysis revealed that both peptides had a molecular mass of ~5.2 kDa and were types of bacteriocins/enterions. Because this bacterium was isolated from nesting mothers and chicks, it is speculated this bacteria helps protect the mother and chicks during the nesting period from microbial infections.

	PROTEOMICS AND AVIAN PARASITES

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
The parasite Eimeria maxima causes coccidiosis in poultry. Two antigens present in the E. maxima’s gametocyte have been identified and are known as M_r 56k Da and M_r 82k Da (Wallach et al., 1989). Proteomic techniques were used to characterize these antigens as potential candidates for a recombinant subunit vaccine (Belli et al., 2002). Two-dimensional PAGE analysis revealed these proteins are acidic but possess a heterogeneous charge. Mass spectrometry analysis gave molecular masses of 52.45 kDa and 62.45 kDa for M_r 56 kDa and M_r 82 kDa, respectively. Mass spectrometry was also used to sequence the N-terminus of each protein, which revealed that there is no homology between these proteins and any other previously identified proteins. It was shown that both proteins also contain O-linked glycans. Proteomic study of these 2 antigenic proteins has revealed biochemical properties of these proteins, which can be useful in designing a vaccine.

	PROTEOMICS AND AVIAN VIRUSES

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
The yeast 2-hybrid system has been widely used in the study of avian infectious diseases. This system has been used to identify potential host receptors for various viruses. It has also been useful to study how viral proteins interact with each other, as well as with other host proteins besides cellular receptors. The yeast 2-hybrid system has been used to look at various virus/host interactions. For example, the 2-hybrid system indicated that the integrase from avian sarcoma retrovirus directly interacts with the host protein Daxx (Greger et al., 2005). Further analysis revealed that Daxx represses viral gene expression. This yeast 2-hybrid screen thus revealed a novel immune response against viral replication.

Infectious bursa disease virus (IBDV) is a member of the birnaviridae family. The virus consists of a dsRNA 2-segment genome and is nonenveloped. Infection with IBDV serotype I results in immunosuppression due to the destruction of B-lymphoid cells and thus represents a major problem for the poultry industry. This virus encodes 5 proteins, 3 nonstructural and 2 structural. The VP1 is the RNA-dependent RNA polymerase, VP2 is structural and makes up the outer layer of the virion, VP3 makes up the inner layer of the virion, VP4 is a viral protease, and VP5 is a nonstructural protein of unknown function, although it is not needed for viral replication. A yeast 2-hybrid screen to study interactions among the viral proteins revealed that with the exception of VP1, all of the IBSV proteins strongly interact with themselves. The VP1 was found to weakly interact with itself. It was also discovered that VP1 and VP3 interact (Tacken et al., 2000, 2003). To determine which domains are involved in these interactions, 5 deletion mutations were created for each protein and assayed by yeast 2-hybrid (Tacken et al., 2003). For VP2 all mutants showed interactions although in varying degrees, indicating that this protein interacts with itself at more than 1 contact site. For VP3, deletion mutants revealed that it interacts with itself at the N-terminal domain and with VP1 at the C-terminal domain. Further deletions revealed that residues 33–129 in the N-terminal domain are involved in VP3-VP3 interactions. The viral protease VP4 proved to be very sensitive with none of the mutations exhibiting interactions. Finally, for VP5 only 1 mutation in the C-terminal domain failed to show self-interaction. Further analysis revealed that residues 73–90 are required for VP5-VP5 interactions, whereas the flanking domains enhance this interaction. The RNase treatments and RT-PCR revealed that VP3 also interacts with both dsRNA segments of the IBDV genome (Tacken et al., 2002).

Marek’s disease virus (MDV) is a herpesvirus that induces Marek’s disease, which is a lymphoproliferative disease in chickens. Marek’s disease is detrimental to the poultry industry resulting in the loss of millions of dollars worldwide. As stated earlier, certain genes, such as MHC haplotypes, have been associated with susceptibility and resistance to MDV. However, susceptibility and resistance to MDV are complex and are governed by many different genes as well as environmental factors. It was found that an infectious clone of MDV, RM1, had lost its oncogenicity and that there was an insertion of a LTR upstream of the MDV SORF2 gene of this clone (Witter et al., 1997). A yeast 2-hybrid screen using a splenic cDNA library and SORF2 revealed an interaction between SORF2 and growth hormone (Liu et al., 2001). This interaction was confirmed by a coimmunoprecipitation assay, and it was also demonstrated via indirect immunoflourescence that SORF2 and growth hormone could be coexpressed in vivo. A genetic study further suggested that growth hormone could contribute to Marek’s disease resistance.

Like other herpes viruses, MDV has a lytic and a latent phase of infection. The switch from the lytic to the latent stage and vice versa is poorly understood, so proteomic profiles of cells lytically infected with MDV and cells in which MDV is latent could reveal what proteins are involved in this switch (Liu et al., 2006). A proteomic profile of MDV lytically infected chicken embryo fibro-blast cells using strong cation exchange chromatography and microcapillary reversed-phase liquid chromatography-tandem mass spectrometry identified 82 expressed MDV proteins, covering almost 80% of MDV proteins. The most frequently sequenced proteins included those involved in DNA replication and capsid formation, which would be expected during productive infection. A proteomic profile of latently infected cells could reveal differences in proteins expressed during both stages.

In addition to yeast 2-hybrid, other methods have been utilized in the study of protein-protein interactions in avian infectious diseases. For example, Chai and Bates (2006) recently used an alternative approach to identify the host receptor of avian leukosis virus subgroup J, which is a retrovirus that induces myeloid leukosis. These investigators identified the receptor by first fusing the surface subunit domain of the viral envelope glycoprotein to the F_c region of rabbit IgG. This fusion protein was then biotinylated and used to precipitate permissive cell surface proteins that interact with the envelope glycoprotein. After SDS-PAGE analysis the interacting proteins were in gel trypsin digested, sequenced via mass spectrometry, and a potential receptor for avian leukosis virus subgroup J, NHE1, was identified.

	CONCLUDING REMARKS

TOP ABSTRACT INTRODUCTION BENEFITS AND LIMITATIONS OF... SYSTEMS BIOLOGY CHICKEN AS A MODEL... PROTEOMICS AND THE EGG PROTEOME OF GERM CELLS PROTEOMICS AND FACIAL... PROTEOMICS AND THE EYE PROTEOMICS AND MUSCLE PROTEOMICS AND THE AVIAN... PROTEOMICS AND AVIAN PARASITES PROTEOMICS AND AVIAN VIRUSES CONCLUDING REMARKS REFERENCES

 
The use of proteomics has greatly increased our knowledge of protein expressions and interactions in avian systems and in avian infectious diseases. However, much about the interactions of proteins to make functioning systems remains to be elucidated. Proteomics has some wonderful aspects that make it very useful in studying systems biology; however, there are some shortcomings that must be overcome. Despite this, proteomic tools are great assets to the study of avian systems biology and infectious diseases.

	FOOTNOTES

 
¹ Presented as part of the Ancillary Scientists Symposium, Functional Genomics: Building the Bridge between the Genome and Phenome, Poultry Science Association Annual Meeting, Sunday, July 16, 2006.

Received for publication February 6, 2007. Accepted for publication February 10, 2007.

	REFERENCES

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