Proteomic analysis of tyrosine phosphorylation during human liver transplantation

Abstract

Background: Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies.

Results: Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R.

Conclusion: Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

Figure 1

Biopsy collection and evaluation of global tyrosine phosphorylation patterns upon I/R. A. Schematic representation of biopsy collection timing illustrating the surgical phases and corresponding liver injury. B. Representative immunoblot analysis of tyrosine phosphorylation on total fractions for 0, 10 and 60 min ischemia (I0, I10, I60) and 0, 10 and 60 min reperfusion (R0, R10, R60) liver protein extracts. N = 2 on 3 independent pools of 3 liver biopsy protein extracts.

Figure 2

Identification of tyrosine phosphorylated proteins upon I/R. A. Schematic representation of the approach used for tyrosine phosphorylated proteins identification. PY matrix: anti-phosphotyrosine antibodies coupled to agarose beads. B. Representative SDS-PAGE experiment after tyrosine immunoprecipitation (Ip PY) of 60 min ischemia (I60) and 60 min reperfusion (R60) protein extracts. The band marked by an arrow has been further identified as the SH₂/SH₃ adaptor Nck-1. N = 1 on 2 independent pools of 3 liver biopsy protein extracts. C. Pie-chart representation of the total number of proteins identified with a significant Mascot score by at least one unique peptide for the 60 min ischemia (I60, left panel) and the 60 min reperfusion (R60, right panel). The proteins were classified in functional families according to their GO (Gene Ontology) annotation.

Figure 3

Nck-1 tyrosine phosphorylation status upon I/R. A. Nck-1 sequence: peptides identified by mass spectrometry are represented underlined, predicted tyrosine phosphorylation site are in red bold. B. Representative MS/MS spectrum corresponding to an Nck-1 specific peptide. C. Prediction of tyrosine-phosphorylation sites using the NetPhos software. D. Immunoblot analysis of Nck-1, using anti-Nck-1 antibody (Ib Nck-1) and Nck-1 tyrosine-phosphorylation status, using anti-phosphotyrosine antibody (Ib PY) following Nck-1 immunoprecipitation (Ip) on 60 min ischemia (I60) and 60 min reperfusion (R60) fractions. The band corresponding to Nck-1 is indicated by a black arrow. Immunoglobulins Heavy Chains (Ig HC) are indicated by a purple arrow. N = 2 on 3 independent pools of 3 liver biopsy protein extracts.

Figure 4

Nck-1 expression and subcellular localization in human liver upon I/R. A. Immunoblot analysis of Nck-1 from total fractions of 0, 10 and 60 min ischemia (I0, I10, I60) and 0, 10 and 60 min reperfusion (R0, R10, R60) liver protein extracts (WCL: Whole Cell Lysate) normalized to Intersectin (Int). N = 2 on 3 independent pools of 3 liver biopsy protein extracts. B. Immunoblot analysis of Nck-1 from insoluble (P100 – lower panel) and soluble (S100 – upper panel) fractions obtained after centrifugation of liver biopsies homogenized in the presence of 150 mM KCl respectively normalized to Ribophorin (Rib) and Intersectin (Int) for the 3 ischemia time points – 0 (I0), 10 (I10) and 60 (I60) min and the 3 reperfusion time points 0 (R0), 10 (R10) and 60 (R60) min. N = 2 on 3 independent pools of 3 liver biopsy protein extracts. C. Immunohistochemical detection of and Nck-1 on 8 μm liver tissue sections 0 and 60 min post-cold ischemia (I0 and I60) and 60 min post-reperfusion (R60). Cells were counterstained with haematoxylin (cells nuclei appear in blue). A representative experiment out of the 6 performed on independent ischemic and reperfused livers is shown. Scale bar = 10 μm.

Figure 5

Determination of Nck-1 interactants by GST pull-down. A. Schematic representation of the approach used for tyrosine mass spectrometry identification of GST Nck-1 binding proteins. GST Nck-1 matrix: GST Nck-1 fusion protein coupled to sepharose beads. B. Representative SDS-PAGE experiment after GST Nck-1 pull-down of 0 (I0) and 60 min ischemia (I60) and 0 (R0) and 60 min reperfusion (R60) protein extract. Bands corresponding to GST Nck-1 fusion protein and cleavage products (Nck-1 and GST) are marked by arrows. N = 1 on 2 independent pools of 3 liver biopsy protein extracts. C. Venn diagram representation of the proteins identified after mass spectrometry analysis of GST Nck-1 binding proteins.