Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 83
Текст из файла (страница 83)
This step includesthe development of cell culture and/or transgenic mouse models to test the function of the protein within the context of the signaling system under examination.This is inherently the most time consuming step of the platform and is also thestep that will vary the most depending on the system and the proteins being studied. Nonetheless, some basic tenets guide the determination of functional rolesfor the identified proteins: 1) assays for the activity of the protein are performed(e.g. kinase activity, phosphor-specific antibodies, and DNA binding) to determinethe effects that association of the given molecule with the subproteome of interest17.3 Metabolic and Transcription/Translation Functions of the PKCe Complexhas on its known activity; 2) inhibitors (e.g.
pharmacological or dominant negativeproteins) of the protein, if available, are used to perturb the function of the protein in vivo and to characterize how the subproteome in focus and the phenotypeare affected; and 3) cell culture or transgenic animal models (e.g.
knockout animals or other transgenic mutants) are created to understand the role of the protein in the given phenotype. This final step of the functional proteomic platformis essential in order to understand the relevance of the observed protein interactions within the context not only of the subproteome, but also with regard to howthey affect the phenotype.17.3Metabolic and Transcription/Translation Functions of the PKCe ComplexOver the past four years, our laboratory has developed and implemented theabove-described platform to characterize PKCe signaling complexes in the myocardium [8, 10]. Previous studies had shown that PKCe was a critical mediator of cardiac protection [16, 17], but the specific mechanism by which PKCe directed thisprotection remained unknown.
We found that PKCe forms complexes with atleast 93 proteins in the mouse heart, and that its association with these proteinsis dynamically regulated by multiple factors [8, 10–13, 20]. We have so far identified at least six distinct classes of molecules in the PKCe subproteome, which are:signaling proteins, stress-activated proteins, structural proteins, metabolism-related proteins, transcription- and translation-related proteins, and PKCe bindingdomain containing proteins [8, 10]. These findings represent a conceptual framework within which many of the salubrious actions of PKCe and cardiac protectioncan be further investigated and understood. These findings are especially noteworthy in that the proteins identified in PKCe complexes are known to accomplish subcellular tasks necessary to prevent cell death during ischemia, therebysuggesting a role for PKCe in modulating these activities.
We hypothesize that byforming distinct modules at different subcellular locations (Fig. 17.2), PKCe directs signal transduction to prevent cell death due to an ischemic insult [9–13]. Inparticular, the rest of the chapter will focus on the possible metabolic and proteinsynthesis regulatory actions of the PKCe complex.Various stimuli that protect the heart from ischemia are known to act in part byimproving cellular metabolism [43–46]. One recent example is a report by Jenningsand colleagues, in which they found that the protected myocardium functions with areduced energy demand [45]. This was measured as a decreased rate of lactate production and a slower depletion of the adenine nucleotide pool in the cardiac muscle.In another example, PKC was shown to be essential to restore high energy phosphate production following ischemia and to prevent glycolytic intermediate accumulation and lactate production [46].
However, the mechanism by which PKC contributes to this enhanced metabolic state during ischemia was unknown.We found that PKCe associates with at least 15 proteins that are involved in theregulation of cellular metabolism [10]. These molecules include glycolytic en-30130217 Proteomic Characterization of Signaling Complexeszymes such as glyceraldehyde phosphate dehydrogenase and enolase, and citricacid cycle enzymes such as succinate dehydrogenase and isocitrate dehydrogenase. These findings implicate PKCe in processes relating to the metabolism offats, carbohydrates and proteins. In addition, we discovered a group of enzymesthat are known to be critically involved in mitochondrial energy balance, including the adenine nucleotide transporter and the voltage-dependent anion channel.These proteins have subsequently been confirmed to be members of the PKCesubproteome by western blotting, and functional assays are underway to understand how association of these molecules with PKCe affects their activity.
The ultimate goal of these functional studies is to fully detail the signaling blueprintthrough which PKCe mediates improved metabolism of the cardiomyocyte, and tounderstand how this contributes to enhanced cell survival during ischemia.Cardiac protection regulated by PKCe has also been previously shown to requireup-regulation of protective proteins [17, 47]. Some of these proteins include iNOSand Src tyrosine kinase, both of which are also members of the PKCe complex [8].In addition, previous studies suggest that cardiac protection requires increasedprotein synthesis [47] and the activation of transcription factors such as AP-1,NFjB, and STAT1 [20, 48, 49].
Despite this, the complete visage of PKCe-dependent signaling to control protein synthesis remained undefined. We found thathistones H1.3 and H4, proteins that participate in chromatin structure, and numerous ribosomal proteins (S3, S18, and S19), reside in PKCe complexes. Theregulation of chromatin structure is a well characterized mechanism to controlgene expression, and the association of PKCe with histones implies that one nuclear role for PKCe may be to influence the DNA-protein interactions in nucleosomes. The multiple ribosomal proteins that appeared in PKCe complexes supportthe concept that PKCe may also regulate protein expression through specific tasksat the ribosome. Notably, we also identified 10 heterogeneous nuclear ribonucleoproteins (hnRNPs), which have been shown to be involved in transcription,mRNA transport and regulation, and in the control of translation [50].
One studycharacterized the ability of PKC isoforms to interact with hnRNP K [51], howevernothing is known regarding the functional consequences of an interaction between any of the hnRNPs and epsilon isoform of PKC. The findings of our studies suggest that PKCe may interact with sundry mRNA processing enzymes in order to specifically control protein expression and possibly splicing and thereby regulate the synthesis of protective proteins to resist cell death due to ischemia.
Accordingly, studies are currently underway to understand the specific manner inwhich PKCe regulates the activity of these proteins to ultimately modulate the expression/post-translational modification of protective proteins in the heart. Thelong term goal of all of these studies is the functional validation of all PKCe complex members. In this manner, the proposed platform will allow for a truly annotated picture of how the PKCe subproteome is involved in protecting the myocardium during ischemic insult.17.5 References17.4ConclusionThe concept of multi-protein complexes as a mechanism for signal transductionis gaining increased support from multiple scientific disciplines. Functional proteomic strategies, like the one described in this chapter, will enable researchers tofurther advance the characterization of these complexes in a thorough and nonbiased manner.
The platform described herein empowers the researcher to movethe investigation “within the complex” and to understand the hierarchy of proteininteractions that results in a phenotype. At the same time, this approach directlylinks signaling network information with organ phenotype. In this regard, theminute alterations that occur in protein complexes at different subcellular locations and in response to different stimuli can be determined directly correlated tothe cellular response. Importantly, the platform is malleable enough to accommodate investigation of virtually any protein of interest.
As a result, the approach described herein facilitates the investigation of how protein complexes regulate physiological phenotype.17.5References1234567N. P. Allen, L. Huang, A. Burlingame,M. Rexach, Proteomic analysis of nucleoporin interacting proteins. J. Biol.Chem. 2001, 276, 29268–29274.A. C. Gavin, et al., Functional organization of the yeast proteome by systematicanalysis of protein complexes.
Nature.2002, 415, 141–147.Y. Ho, et al., Systematic identification ofprotein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature.2002, 415, 180–183.H. Husi, M. A. Ward, J. S. Choudhary,W. P. Blackstock, S. G. Grant, Proteomic analysis of NMDA receptor-adhesionprotein signaling complexes. Nat.
Neurosci. 2000, 3, 661–669.J. D. Jordan, E. M. Landau, R. Iyengar,Signaling networks: the origins of cellularmultitasking. Cell. 2000, 103, 193–200.Y. Liu, J. A. Ranish, R. Aebersold, S.Hahn, Yeast nuclear extract contains twomajor forms of RNA polymerase II mediator complexes. J. Biol. Chem. 2001, 276,7169–7175.G. Neubauer, J. Rappsilber, C. Calvio,M. Watson, P.
Ajuh, J. Sleeman, A. La-891011mond, M. Mann, Mass spectrometry andEST-database searching allows characterization of the multi-protein spliceosomecomplex. Nat. Genet. 1998, 20, 46–50.P. Ping, J. Zhang, W. M. Pierce, Jr., R.Bolli, Functional proteomic analysis ofprotein kinas C e signaling complexes inthe normal heart and during cardioprotection. Circ. Res. 2001, 88, 59–62.P.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
