The recent genomic revolution has resulted in a paradigm drift in the comprehensive analysis of biological processes and biochemical regulators. A continuous body of literature strongly indicating a non-predictive correlation between mRNA and protein level expression [28
], along with posttranscriptional modifications leading to control in the rate of synthesis and half-life of proteins place [29
] an increased importance on the direct measurement of the protein expression by proteomic techniques to analyze the biochemical signaling pathways. In this report using various proteomics based approaches we determine the key role of regulatory molecular chaperonins in the I/R injury in the setting of liver steatosis.
The demand for liver allografts remains a challenging problem for transplant centers in the US and across the globe, especially with the increasing prevalence of hepatic steatosis in the donor population [30
]. Attenuation of steatosis-associated ischemia/reperfusion injury is an important strategy for reducing allograft shortages. Proteomic analysis is an important tool for large-scale analysis of cellular changes induced through various injury models, including that of hepatic ischemia . This particular analysis revealed that the proteome profile of steatotic liver following a period of warm ischemia/reperfusion significantly differs from that of lean liver. Some functional groups, such as those involved with amino acid metabolism, fatty acid metabolism, and cellular structure, are expected to be significantly different in steatotic liver, regardless of I/R injury. A novel finding, however, was the significant down-regulation of multiple molecular chaperones. Chaperonins have important roles in protein folding and structure [31
]. Disruptions in cellular homeostasis can cause perturbations in protein processing, resulting in an accumulation of unfolded proteins. This triggers the “unfolded protein response” (UPR), or endoplasmic reticulum (ER stress) response, a response to cellular stress which can lead to adaptation or trigger apoptotic pathways [32
]. Chaperonins reduce ER stress by stabilizing proteins which become unfolded during periods of cellular stress (e.g.
, I/R). Pdia3 encodes ERp57, a known molecular chaperone with well-established interactions with calreticulin and their role in antigen-processing/loading of MHC class I molecules [33
]. ERp57 is also known to regulate Grp78, a key molecular chaperone in initiating one of three ER stress signaling cascades [35
]. The chaperonin subset of proteins, with these molecules in particular, represent potential therapeutic targets for mitigating I/R injury.
The Akt pathway, a serine/threonine kinase, has been shown to play an important role in various types of ischemia/reperfusion. Although it did not show significant differential expression at the reperfusion time points analyzed in our study, Akt is indirectly related to multiple chaperonins which do play a significant role in protein folding and processing, and have implicated roles in endoplasmic reticulum stress in other models. Akt has also been shown to play a role in ER stress. Given these relationships which we have identified, it is likely that ER stress plays a role in the greater injury sustained by steatotic liver following intervals of ischemia/reperfusion [36
]. Also, certain key mediators of the unfolded protein response (Grp78, IRE-1a, XBP-1(s), ATF4, and CHOP) are shown to be upregulated in both a murine model of hepatic warm I/R and a rat liver transplant model [37
The potential role of chaperones as drug targets has been well established. Previous studies in our laboratory using a chaperones targeting small chemical molecule, taurine-conjugated ursodeoxycholic acid (TUDCA), has shown to decrease ER stress response and decrease ischemia repurfusion injury in animal models of steatotic liver allografts [38
]. The role of chaperones in ischemia injury has also been established in other transplant models such as cardiac myocytes [39
]. Not just limited to transplant settings, but in other disease states such as malaria, chaperones are considered as important drug targets [40
]. However, in our initial study along with chaperones/ER stress protein group we have also identified other protein groups such as oxidative stress, metabolism, and cell structure. Although, it is currently unclear of their potential role in drug targeting these protein groups in ameliorating I/R injury and enhancing survival of steatotic liver allografts, future research on these protein groups could offer novel molecular targets.
Certain inherent limitations with 2-DE based proteomics approaches associated with artifacts and database subset mapping in identifying exact target proteins [41
] warrants further direct cell based functional studies to validate our current findings. Lean liver tissue served as the comparison, or control, for steatotic liver subjected to warm ischemia and a short period of reperfusion; however, it might be revealing to compare these samples to liver tissue not subject to I/R. Samples obtained at additional reperfusion times would help establish a trend in protein expression profiles. In spite of limitations we still consider our current study to add new insights and further research to study the functional basis and potential chaperonin-based therapeutic strategies to enhance long term survival of the liver allografts.