Browsing by Subject "proteasome"

Now showing 1 - 6 of 6
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    Assembly of proteasome subunits into non-canonical complexes in vivo
    (Elsevier, 2017-01) Hammack, Lindsay J.; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    Proteasomes exist in all domains of life. In general, they are comprised of a compartmentalized protease whose activity is modulated by one or more regulatory complexes with which it interacts. The quaternary structure of this compartmentalized protease, called the 20S proteasome, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the α and β subunits during 20S biogenesis. Here we demonstrate that proteasome subunits can assemble into structures other than the canonical 20S proteasome in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring-shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non-canonical complexes exist in vivo.
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    A Conserved 20S Proteasome Assembly Factor Requires a Cterminal HbYX Motif for Proteasomal Precursor Binding
    (2011-05) Kusmierczyk, Andrew R; Kunjappu, Mary J; Kim, Roger Y; Hochstrasser, Mark
    Dedicated chaperones facilitate the assembly of the eukaryotic proteasome, but how they function remains largely unknown. Here we show that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal hydrophobic-tyrosine-X (HbYX) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX motif–dependent fashion using the same proteasomal α-ring surface pockets as are bound by activators. PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data are consistent with an allosteric mechanism in which the maturation of the proteasome active sites determines the binding of assembly chaperones, potentially shielding assembly intermediates or misassembled complexes from nonproductive associations until assembly is complete.
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    Evidence for a regulatory role of Cullin-RING E3 ubiquitin ligase 7 in insulin signalling
    (Elsevier B.V., 2014-02) Scheufele, Florian; Wolf, Benjamin; Kruse, Michael; Hartmann, Thomas; Lempart, Justine; Mühlich, Susanne; Pfeiffer, Andreas F. H.; Field, Loren J.; Charron, Maureen J.; Pan, Zhen-Qiang; Engelhardt, Stefan; Sarikas, Antonio; Department of Medicine, IU School of Medicine
    Dysfunctional regulation of signalling pathways downstream of the insulin receptor plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. In this study we report both in vitro and in vivo experimental evidence for a role of Cullin-RING E3 ubiquitin ligase 7 (CRL7) in the regulation of insulin signalling and glucose homeostasis. We show that Cul7−/− mouse embryonic fibroblasts displayed enhanced AKT and Erk MAP kinase phosphorylation upon insulin stimulation. Depletion of CUL7 by RNA interference in C2C12 myotubes led to increased activation of insulin signalling pathways and cellular glucose uptake, as well as a reduced capacity of these cells to execute insulin-induced degradation of insulin receptor substrate 1 (IRS1). In vivo, heterozygosity of either Cul7 or Fbxw8, both key components of CRL7, resulted in elevated PI3 kinase / AKT activation in skeletal muscle tissue upon insulin stimulation when compared to wild-type controls. Finally, Cul7+/− or Fbxw8+/− mice exhibited enhanced insulin sensitivity and plasma glucose clearance. Collectively, our findings point to a yet unrecognized role of CRL7 in insulin-mediated control of glucose homeostasis by restraining PI3 kinase / AKT activities in skeletal muscle cells.
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    Identifying Potential Proteasomal assembly factors and/or binding proteins using the yeast Saccharomyces cerevisiae as a model organism
    (Office of the Vice Chancellor for Research, 2015-04-17) Lindsay, Nicole; Hammack, Lindsay; Kusmiercyzk, Andrew
    The proteasome is a large multi-protein complex responsible for the ultimate degradation of proteins in the cell. Damaged or misfolded proteins are targeted for destruction and broken down into peptides. Proteasomal degradation plays a vital role in almost every cellular process, from the cell cycle, to cell development, to apoptosis. Moreover, understanding and identifying the proteasome assembly process, important binding factors, and chaperones that assist in proteasome assembly would be pivotal in developing strategies to remedy cellular disorders caused by defects in proteasomal function. The eukaryotic proteasome is composed of two main sub-complexes, a 20S core particle and a 19S regulatory particle that caps one or both ends of the 20S core particle. The 20S core particle is the degradation component of the proteasome, and it is made up of 14 unique subunits with seven distinct α and β subunits that assemble into four stacked heteroheptameric rings. On the β7 subunit, there is a C-terminal peptide tail that connects two halves of the 20S core particle. Previous research has shown that deletion of the β7 tail slows down proteasome assembly. We generated a yeast strain containing a deletion of the β7 tail along with deletion of two assembly factors, Pba1p and Ump1p. This strain is severely temperature sensitive and will be used to screen a plasmid-borne yeast genomic library. The goal is to potentially identify new proteasomal chaperones and/or binding partners which, when present in high copy, can overcome the defect imposed by the triple mutant.
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    Molecular chaperones of the Hsp70 family assist in the assembly of 20S proteasomes
    (Elsevier, 2017-04) Hammack, Lindsay J.; Firestone, Kyle; Chang, William; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    The eukaryotic 26S proteasome is a large protease comprised of two major sub assemblies, the 20S proteasome, or core particle (CP), and the 19S regulatory particle (RP). Assembly of the CP and RP is assisted by an expanding list of dedicated assembly factors. For the CP, this includes Ump1 and the heterodimeric Pba1–Pba2 and Pba3–Pba4 proteins. It is not known how many additional proteins that assist in proteasome biogenesis remain to be discovered. Here, we demonstrate that two members of the Hsp70 family in yeast, Ssa1 and Ssa2, play a direct role in CP assembly. Ssa1 and Ssa2 interact genetically and physically with proteasomal components. Specifically, they associate tightly with known CP assembly intermediates, but not with fully assembled CP, through an extensive purification protocol. And, in yeast lacking both Ssa1 and Ssa2, specific defects in CP assembly are observed.
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    Temperature Sensitive Mutant Proteome Profiling (TeMPP) A Tool for the Characterization of Global Impacts of Missense Mutations on the Proteome
    (2020-07) Justice, Sarah Ann; Mosley, Amber L.; Harrington, Maureen A.; Goebl, Mark G.; Bidwell, Joseph P.
    Thousands of missense mutations have been found to be associated with human diseases, ~60% of which have been predicted to affect protein stability and/or protein-protein interactions (PPIs). Current proteomic methods for studying the effects of mutations on the cell focus on measures of protein abundance or post-translational modifications (PTMs), which cannot directly be used for PPI analysis. High-throughput methodology to evaluate how mutations in a single protein affect PPI networks would help streamline the characterization of global effects caused by mutant proteins and aid in the prediction of phenotypic outcomes resulting from genomic mutations. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of a mass spectrometry (MS) based thermal proteome profiling (TPP) approach that measures changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. This study measures the impact of temperature sensitivity-inducing missense mutations of proteins in the ubiquitin proteasome system and the transcription termination machinery on the thermal stability of the proteome at large. Results reveal distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. Furthermore, my data suggests that TeMPP is highly specific to proteins functionally related to the mutated protein of interest and capable of differentiating effects between two proteins in the same complex. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion. Use of this method along with other complementary -omics approaches will help to characterize how missense mutations affect cellular protein homeostasis and thus enable deeper insight into disease phenotypes.