Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Øverbye, Anders; Fengsrud, Monica; Seglen, Per Ottar

doi:10.4161/auto.3910

Cited by 143 publications

(125 citation statements)

References 196 publications

(251 reference statements)

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“…Overbye et al reported that 39 proteins significantly enriched (by 2Â or more) in autophagosomal membranes prepared from rat hepatocytes treated with vinblastine, an microtubule-disrupting drug that blocks the fusion of hepatocellular autophagosomes with lysosomes, were identified in proteomic analysis. 34) It is interesting that the TG protein substrate candidates (AI, FBPase, BHMT, GAPDH, and GST) identified in our studies, present and previous, are all found in the 39 proteins enriched in the autophagosomal membranes. Moreover, BHMT fragments (p32 and p35) have been found to accumulate in mitochondrial-lysosomal subcellular fractions and autolysosomes from rat hepatocytes treated with leupeptin, an inhibitor of intralysosomal protein degradation.…”

Section: Discussionmentioning

confidence: 86%

Identification of New Amine Acceptor Protein Substrate Candidates of Transglutaminase in Rat Liver Extract: Use of 5-(Biotinamido) Pentylamine as a Probe

Ichikawa

Ishizaki

Morita

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Transglutaminases (TGs) are a family of enzymes that catalyze Ca 2þ -dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40-and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.

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Section: Discussionmentioning

confidence: 86%

Identification of New Amine Acceptor Protein Substrate Candidates of Transglutaminase in Rat Liver Extract: Use of 5-(Biotinamido) Pentylamine as a Probe

Ichikawa

Ishizaki

Morita

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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“…7 Much of the pioneering work in the autophagy field is conducted in the liver or using isolated hepatocytes, such as determination of the morphology and membrane compositions of autophagosomes and characterization of the kinetics of protein degradation, regulation by hormones and amino acids, and involvement of the mammalian target of rapamycin (mTOR) pathway in induction. 2,[8][9][10][11][12] It is now clear that macroautophagy is important for many physiological and pathological processes. 4,6,13 It is required for normal development and participates in the clearance of apoptotic cells during embryogenesis.…”

Section: The Basics Of Macroautophagymentioning

confidence: 99%

Autophagy in the liver

2008

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A great part of our current understanding of mammalian macroautophagy is derived from studies of the liver. The term "autophagy" was introduced by Christian de Duve in part based on ultrastructural changes in rat liver following glucagon injection. Subsequent morphological, biochemical, and kinetics studies of autophagy in the liver defined the basic process of autophagosome formation, maturation, and degradation and the regulation of autophagy by hormones, phosphoinositide 3-kinases, and mammalian target of rapamycin. It is now clear that macroautophagy in the liver is important for the balance of energy and nutrients for basic cell functions, the removal of misfolded proteins resulting from genetic mutations or pathophysiological stimulations, and the turnover of major subcellular organelles such as mitochondria, endoplasmic reticulum, and peroxisomes under both normal and pathophysiological conditions. Disturbance of autophagy function in the liver could thus have a major impact on liver physiology and liver disease. (HEPATOLOGY 2008;47: 1773-1785.)

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“…Existing evidence indeed support such a scenario. For example, it has been shown that BHMT is highly enriched on autophagosomal membranes, 51,52 suggesting a potential direct or indirect physical interaction with autophagosome proteins including MAP1LC3. Further, cargo receptors SQSTM1 and NBR1 not only recognize and bind to ubiquitinated proteins, but are also required for their assembly into aggregate-like structures (aggresomes) that are important for their subsequent recognition and sequestration by the MAP1LC3-containing autophagosome structures, as supported by the findings that loss of SQSTM1 significantly suppresses the accumulation of ubiquitinated proteins as well as the formation of inclusion bodies both in autophagy-deficient neurons and hepatocytes, and also in hepatocytes with impaired proteasome activity that otherwise show elevated levels of ubiquitinated aggregates.…”

Section: Different Mechanisms Mediate Starvation-and Proteasome Inhibmentioning

confidence: 99%

“…53,54 Given these observations, one putative scenario is that upon proteasomal impairment, cargo receptors SQSTM1 and NBR1 recruit ubiquitinated proteins into aggresomes, a prerequisite step for their recognition by the MAP1LC3-containing membranes that subsequently assemble into mature autophagosomes. During this process, as BHMT are normally highly enriched on the membranes of autophagosomes or their precursors, 51,52 they are passively packaged into autophagosomes and subsequently processed. In the absence of the cargo receptors, aggresomes cannot form, 52,53 thereby abolishing the MAP1LC3-mediated autophagosome formation and the parallel sequestration of BHMT.…”

Section: Different Mechanisms Mediate Starvation-and Proteasome Inhibmentioning

confidence: 99%

“…During this process, as BHMT are normally highly enriched on the membranes of autophagosomes or their precursors, 51,52 they are passively packaged into autophagosomes and subsequently processed. In the absence of the cargo receptors, aggresomes cannot form, 52,53 thereby abolishing the MAP1LC3-mediated autophagosome formation and the parallel sequestration of BHMT. Many other possibilities exist and many questions remain, including whether and how the multimeric BHMT are preferentially associated with autophagosome membranes, that need to be further addressed in the future.…”

Section: Different Mechanisms Mediate Starvation-and Proteasome Inhibmentioning

confidence: 99%

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The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

Rui

Chen

et al. 2015

Autophagy

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#These authors equally contributed to this work.Keywords: BHMT, cargo receptors SQSTM1/p62 and NBR1, MTOR, PRKAA/AMPK, proteasome inhibition, selective macroautophagyAbbreviations: ACACA/B, acetyl-CoA carboxylase a/b; ACTB, actin, b; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; ATG7, autophagy-related 7; Baf A1, bafilomycin A 1 ; BCL2, B-cell CLL/lymphoma 2; BECN1, Beclin 1, autophagy-related; BHMT, betaine-homocysteine S-methyltransferase; CTNNB1, catenin (cadherin-associated protein), b 1, 88kDa; Cvt, cytoplasm-to-vacuole-targeting; DDIT3, DNA-damage-inducible transcript 3; EBSS, Earle's Balanced Salt Solution; EIF2AK3, eukaryotic translation initiation factor 2-a, kinase 3; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; ER, endoplasmic reticulum; ERN1, endoplasmic reticulum to nucleus signaling 1; GST, glutathionine S-transferase; GST-BHMT, a fusion protein of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; GST-BHMT (FRAG) , an autophagy-mediated cleavage product of the GST-BHMT reporter; HA, hemagglutinin; HSPA5, heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa); LSCS, linker-specific cleavage site; MAP1LC3, microtubule-associated protein 1 light chain 3; MAP2K7, mitogen-activated protein kinase kinase 7; MAPK8, mitogen-activated protein kinase 8; MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC1, MTOR complex 1; NBR1, neighbor of BRCA1 gene 1; P4HB, prolyl 4-hydroxylase, b polypeptide; PRKAA, protein kinase, AMP-activated, a catalytic subunit; RHEB, Ras homolog enriched in brain; RM, rich medium; RPS6KB1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; SQSTM1, sequestosome 1; TSC1/2, tuberous sclerosis 1/2; ULK1, unc-51 like autophagy activating kinase 1; UPR, unfolded protein response; UPS, ubiquitin proteasome system; XBP1, X-box binding protein 1By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, a catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic ...

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Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Cited by 143 publications

References 196 publications

Identification of New Amine Acceptor Protein Substrate Candidates of Transglutaminase in Rat Liver Extract: Use of 5-(Biotinamido) Pentylamine as a Probe

Identification of New Amine Acceptor Protein Substrate Candidates of Transglutaminase in Rat Liver Extract: Use of 5-(Biotinamido) Pentylamine as a Probe

Autophagy in the liver

The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

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