A Protein Interaction Network for Ecm29 Links the 26 S Proteasome to Molecular Motors and Endosomal Components

Gorbea, Carlos; Pratt, Gregory; Ustrell, Vicença; Bell, Russell; Sahasrabudhe, Sudhir; Hughes, Robert E.; Rechsteiner, Martin

doi:10.1074/jbc.m110.154120

Cited by 74 publications

(83 citation statements)

References 103 publications

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“…The proteasome‐associated protein ECM29 was identified to be down‐regulated with increasing specific productivity in both groups. It is believed that ECM29 serves as an adaptor for coupling 26 S‐proteasomes to specific cellular compartments (Gorbea et al, 2010). Summarizing the observed results, an increase of lysosomal proteins and a decrease of proteasome associated proteins with increasing specific productivities has been shown.…”

Section: Discussionmentioning

confidence: 99%

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Sommeregger

Mayrhofer

Steinfellner

et al. 2016

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Sommeregger

Mayrhofer

Steinfellner

et al. 2016

Biotech & Bioengineering

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“…USP14 is a DUB, which also regulates the degradative capacity of the 26S (6,39). In yeast, Ecm29 was found to stabilize the 19S-20S association (40), and in mammalian cells, Ecm29 is largely found on vesicular structures of the endosomal pathway (41,42). PA200 (Blm10 in yeast) can form hybrid 26S proteasomes, 19S-20S-PA200, and has been implicated in DNA repair (43).…”

Section: Muscle 26s Proteasomes From Aged and Adult Rats Have A Similmentioning

confidence: 99%

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Altun

Besche

Overkleeft

et al. 2010

Journal of Biological Chemistry

192

155

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Among the hallmarks of aged organisms are an accumulation of misfolded proteins and a reduction in skeletal muscle mass ("sarcopenia"). We have examined the effects of aging and dietary restriction (which retards many age-related changes) on components of the ubiquitin proteasome system (UPS) in muscle. The hindlimb muscles of aged (30 months old) rats showed a marked loss of muscle mass and contained 2-3-fold higher levels of 26S proteasomes than those of adult (4 months old) controls. 26S proteasomes purified from muscles of aged and adult rats showed a similar capacity to degrade peptides, proteins, and an ubiquitylated substrate, but differed in levels of proteasome-associated proteins (e.g. the ubiquitin ligase E6AP and deubiquitylating enzyme USP14). Also, the activities of many other deubiquitylating enzymes were greatly enhanced in the aged muscles. Nevertheless, their content of polyubiquitylated proteins was higher than in adult animals. The aged muscles contained higher levels of the ubiquitin ligase CHIP, involved in eliminating misfolded proteins, and MuRF1, which ubiquitylates myofibrillar proteins. These muscles differed from ones rapidly atrophying due to disease, fasting, or disuse in that Atrogin-1/MAFbx expression was low and not inducible by glucocorticoids. Thus, the muscles of aged rats showed many adaptations indicating enhanced proteolysis by the UPS, which may enhance their capacity to eliminate misfolded proteins and seems to contribute to the sarcopenia. Accordingly, dietary restriction decreased or prevented the aging-associated increases in proteasomes and other UPS components and reduced muscle wasting.

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“…Similar abnormal function of the proteasome can be predicted during impairment of axonal transport because kinesin-1 defects enhance protein accumulation, axonopathies and neurodegeneration in mouse models of Alzheimer's disease (Falzone et al, 2009;Stokin et al, 2005). Recently, a link between proteasomes and motors in mammalian cells has been suggested to occur through the proteasome-associated protein ECM29, which is involved in proteasome assembly (Gorbea et al, 2010). This interaction indicates a molecular mechanism for motor-protein-dependent transport of the proteasome complex.…”

Section: Introductionmentioning

confidence: 99%

Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function

Otero

Alloatti

Cromberg

et al. 2014

Journal of Cell Science

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Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

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A Protein Interaction Network for Ecm29 Links the 26 S Proteasome to Molecular Motors and Endosomal Components

Cited by 74 publications

References 103 publications

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function

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