NeuCode Proteomics Reveals Bap1 Regulation of Metabolism

Abstract

SUMMARY We introduce neutron-encoded (NeuCode) amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode we characterize an inducible knock-out mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in the liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1-null mice present with fully penetrant perinatal lethality, severe hypoglycemia and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in the liver and pancreas, and establishes NeuCode as a reliable proteomic method for deciphering in vivo biology.

BAP1, a tumor suppressor gene driving malignant mesothelioma

“…Quantitative proteomic analysis of tissues from inducible Bap1 KO mice revealed a role in metabolic homeostasis in the pancreas and liver (55). Elevated cholesterol biosynthesis but reduced expression of gluconeogenic and lipid homeostasis proteins were observed in the liver.…”

“…In the pancreas, expression of pancreatitis protein markers was increased whereas expression of mitochondria proteins was decreased. These mice also exhibited hypercholesterolemia, hypoglycemia, lipid reduction in the liver, and pancreatic acinar cell degeneration (55). How such metabolic dysregulation is related to cancer predisposition or tumorigenesis is yet to be determined.…”

Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice

“…Because incorporation rates differ by tissue, it is important to confirm sufficient incorporation time for the tissue of interest. We found that incorporation of isotopologue into liver is faster than into brain or muscle and that 2 weeks of isotopologue incorporation would be sufficient for studying the liver while 4 weeks of incorporation is needed for studying brain or muscle 21 . Experimental models where growth or protein synthesis is severely affected are likely not good candidates for NeuCode labeling approaches for quantitation of relative protein abundance.…”

“…Since 2013, NeuCode SILAC has been applied to a number of different experimental models including yeast 4,11 , adherent cell lines 19 , nematodes 20 , and mice 21 . In this protocol we highlight the use of NeuCode for adherent cell lines and mouse models; however, application of the method to other model systems is straight forward and simply requires the dietary replacement of normal lysine with NeuCode lysine isotopologues.…”

“…Thus, metabolic labeling in primary cells becomes feasible (since multiple generations of labeling are not required), and metabolic labeling in animals becomes faster and more economical. For example, NeuCode labeling of mice can be done with ~ 2-4 weeks of feeding 21 compared to the traditional stable isotope labeled amino acids in mice (SILAM), which requires several generations of isotopologue feeding 24 . Note a caveat of incomplete labeling is that the MS 1 spectral complexity is increased due to the presence of unlabeled peptides.…”