title: PiZ mouse liver accumulates polyubiquitin conjugates that associate with catalytically active 26S proteasomes.
abstract: Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Haddock
authors.affiliation: Department of Pediatrics, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America.
authors.forename: Keith
authors.initials: K
authors.lastname: Blomenkamp
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America.
authors.forename: Madhav
authors.initials: M
authors.lastname: Gautam
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America.
authors.forename: Jared
authors.initials: J
authors.lastname: James
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America.
authors.forename: Joanna
authors.initials: J
authors.lastname: Mielcarska
authors.affiliation: School of Biological Sciences, University of Missouri - Kansas City, Kansas City, Missouri, United States of America.
authors.forename: Edward
authors.initials: E
authors.lastname: Gogol
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America; Department of Pediatrics, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Teckman
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America.
authors.forename: Dorota
authors.initials: D
authors.lastname: Skowyra
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D050505:Mutant Proteins; D000515:alpha 1-Antitrypsin; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases