Subcellular Proteomic Analysis Of Rat Liver Mitochondrial Proteins During Liver Regeneration

Liver is one of the most important organs involved in physiological,pathological,and toxicological events.It is well known that the liver has the capacity to regenerate and restore its original size and function after partial hepatectomy(PHx),injury and hepatic diseases. Liver regeneration continues to be an important focus in mammalian liver research. Acceleration of liver regeneration could be of great clinical benefit in various liver-associated diseases.However,at present little is known about therapeutic interventions to enhance this regenerative process.Our limited understanding and the complexity of the mechanisms involved have prevented the identification of new targets for treatment.Previous studies have identified many pathways involved in this process and have charted the time course of major regulating events that control the replication of hepatocytes and enable this large parenchymal organ to restore its functional integrity after pronounced damage or volume loss.However,the molecular mechanism of liver regeneration,especially the mechanism of priming and termination after partial hepatectomy was not clear yet.In the past years,major investigations were focused on the changes in the gene expression pattern,and little was known about the protein expression pattern of liver regeneration. Proteomics is the large-scale study of proteins,particularly their structures and functions, which provide a very effective approach to explore differentially expressed protein during the liver regeneration.Proteins are central to our understanding of cellular function and disease processes,and without a concerted effort in proteomics,the fruits of genomics will go unrealized.Differential proteomics,the comparison of distinct proteomes(eg,normal versus diseased cells,diseased versus treated cells,etc) is of paramount importance.At the same time, subcellular fractionation and purification of organelles provide attractive additions to protein separation techniques commonly used in proteomic analysis.There has been a tendency to focus on subcellular proteomes concerning specific subcellular compartments and macromolecular structures of the cell.Subcellular proteomics research cannot only provide information about subcellular location of certain protein and imply its function,but also tell us the whole-protein components of the specific subcellular fraction(organelle or other multiprotein complex) and then help understand their structures as well as biological functions.Mitochondria are one of the most complex subcellular organelles and play key roles in many cellular functions including energy production,fatty acid metabolism,calcium homeostasis,and cell signaling.Liver regeneration is a very complicated biological procedure which involves various signal transduction pathways and molecular events.Mitochondria were investigated as they are directly involved in the process of liver regeneration.However, the role of mitochondria during liver regeneration is not clear and a global alteration of mitochondrial protein expression during liver regeneration was not investigated.PHx of approximately 70%is widely used for studies on liver regeneration,hepatocyte differentiation,and regulation of gene or protein expression.The present study was designed to analyze alterations in the mitochondrial proteome in rat liver regeneration after 70%PHx via the classic experimental model,and to find novel significant proteins which correlate with liver regeneration.This proteomic model might add helpful clue to the current knowledge of liver regeneration and might contribute to understanding the events that may occur in mitochondria during liver regeneration in a eukaryote-wide range.Sectionâ… :Differential proteomies analysis of the mitochondrial proteins in rats during liver regeneration after partial hepatectomyIn this study,liver tissues were gained from partial hepatectomy(PHx),a classic experimental model of rapid liver cell proliferation.A purified mitochondrial fraction were obtained from Nycodenz density gradient centrifugation.The proteomic changes in rat liver mitochondria were analyzed in two groups,70%PHx test group and sham-operation control group,at 24 h,72 h and 168 h after 70%PHx.By a modified two-dimensional gel electrophoresis,the differential protein spots were recognized using PDQuest7.4 software. They were identified by matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight mass spectrometry(MALDI-TOF/TOF MS) and/or tandem mass spectrometry reconfirmation.The data from MALDI-TOF/TOF MS were analyzed by using the program MASCOT v.1.9 against SWISS-PROT database with GPS explorer software.To confirm these results,various important identified proteins were validated by real-time PCR and Western blotting.We found twenty-two down-regulated spots and three up-regulated spots in mitochondria at 24 h after 70%PHx.In all twenty-two differentially expressed proteins were successfully identified which were associated with carbohydrate metabolism,lipid metabolism,respiratory chain and oxidation-phosphorylation,biotransformation and other metabolic pathways.These results indicate that liver regeneration following partial hepatectomy affects various metabolic pathways and advance knowledge of the role of mitochondria during the early phase of liver regeneration.In agreement with an earlier report, our results suggested a dysfunction of mitochondria at 24 hours after 70%PHx,which may be involved in causing the lag of liver regeneration at this phase.It is a plausible,albeit incomplete,suggestion from others and our data that mitochondria might have a preparing role in the early phase of liver regeneration,and thereafter it has an important role in remodeling of the liver tissue.Sectionâ…¡:Investigation of the potential cellular function of prohibitin(PHB) during liver regenerationProhibitin,a potential tumor suppressor protein,have been ascribed various functions, such as cell cycle regulation,senescence,transcription regulation,tumor suppression and apoptosis.But up to date,most studies focused on the role of prohibitin in some kinds of tumors and little is known about its role during liver regeneration.By a subcellular proteomics analysis we found prohibitin was down-regulated in mitochondria at 24 h after 70%PHx.Liver regeneration is a very complicated biological procedure which involves various signal transduction pathways and molecular events.Thus, we thought that prohibitin may have a crucial role during liver regeneration and chosen this protein for further investigations.To investigate the potential cellular function of prohibitin during liver regeneration and its association with mitochondria,the changes of prohibitin expression,subcellular distribution and mitochondrial alterations during liver regeneration were observed.In vivo,comparing with sham-operation control group,we found prohibitin mRNA and protein expression showed concordant changes during liver regeneration after 70%PHx:both reduced at 24 h,slightly up-regulated at 72 h and evidently increased at 168 h.We found prohibitin was mainly located in mitochondria in rat livers,and its abundance was down-regulated at 24 h,evidently increased at 72 h and nearly recovered to normal at 168 h after 70%PHx.Interestingly,mitochondria showed a marked changes in ultrastructure at 24 h, a slight changes at 72 h and recovered nearly to normal at 168 h after 70%PHx.The reduced mitochondrial mass also nearly recovered to normal at 168 h after 70%PHx.Prohibitin also showed in nucleus with an increased protein levels during liver regeneration.No prohibitin was found in cytosol.In vitro,knockdown of prohibitin by siRNA or prohibitin overexpression in BRL-3A cells,the results all showed that:prohibitin has a role in maintaining mitochondrial membrane potential,which might affect mitochondrial function;and prohibitin can prevent the cell cycle entry from G1 to S;and it appears that prohibitin also has anti-apoptotic effects.In combination with previous reports,we suggest that prohibitin might regulate cell proliferation during liver regeneration in a complex manner,which could involve mechanisms mediated by both mRNA and protein.And our data suggest that prohibitin has a dual function during liver regeneration:one is as a negative regulator of cell cycle for the homeostasis and keeping the balance between proliferation and apoptosis,and the other,in modulating and maintaining mitochondrial stabilization.It is not yet known whether the dual function is directly linked to each other or represents two independent mechanisms.Further study will help us to uncover the complexity and function of prohibitin during liver regeneration and targeting prohibitin might provide an useful therapeutic approach for the treatment of liver-associated diseases.To our knowledge,this is the first description of mitochondrial proteome alterations after surgical reduction of liver volume that have been identified by 2-DE in combination with MS, and protein identification via PMF and MS/MS.Subcellular proteomic analysis of mitochondrial proteins in the early phase of liver regeneration provided an effective approach for better understanding the role of mitochondria during liver regeneration.The changes in the expression of several proteins that we have documented after PHx reflect the involvement of various cellular metabolic pathways.Our results point to several cellular functions,not yet studied in the context of liver regeneration,as valuable targets for further investigation. Prohibitin was shown to be involved in the maintenance of mitochondrial stabilization,and keeping the balance between proliferation and apoptosis,which might provide an approach to understanding the mechanism underlying the function of mitochondria during liver regeneration.The role of prohibitin in the process of liver regeneration needs further studies.