Catalytic Properties of 26 S and 20 S Proteasomes and Radiolabeling of MB1, LMP7, and C7 Subunits Associated with Trypsin-like and Chymotrypsin-like Activities

Reidlinger, Jutta; Pike, Angela M.; Savory, Peter; Murray, Rachael Z.; Rivett, A. Jennifer

doi:10.1074/jbc.272.40.24899

Cited by 61 publications

(51 citation statements)

References 54 publications

(66 reference statements)

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“…This unstructured protein has been shown to be a good substrate for both the 20S and 26S proteasome (Reidlinger et al, 1997). Comparing the rates of degradation, we found a significant ~40% decrease in the rate of casein degradation for proteasome modified by 2 μM NEM ( Figure 5).…”

Section: Degradation Of Protein Substratesmentioning

confidence: 86%

“…Association of the regulatory complexes, PA28 and PA700, with the 20S catalytic core has been shown to increase proteasome activity (DeMartino et al, 1996;Dubiel et al, 1992;Ferrington et al, 2005;Reidlinger et al, 1997). Using antibodies that recognize the α-subunit of PA28 and the S4 subunit of PA700, we evaluated the content of these proteasome activators in the retina and RPE.…”

Section: Content Of Regulatory Complexesmentioning

confidence: 99%

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Age-dependent inhibition of proteasome chymotrypsin-like activity in the retina

Kapphahn

Bigelow

Ferrington

2007

Experimental Eye Research

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The proteasome plays a fundamental role in processes essential for cell viability. A loss in proteasome function has been associated with aging, as well as a number of age-related diseases. Defining the mechanism(s) behind this loss in function will add important information regarding the molecular basis for aging. In the current study, we performed an age-based comparison of proteasome function and composition of subunits and regulatory proteins in the neural retina and retinal pigment epithelium (RPE) in Fischer 344 rats. In the RPE, there was no age-dependent difference in activity, subunit composition, or content of proteasome regulators, PA28 and PA700. In contrast, the aged neural retina demonstrated a significant reduction in the chymotrypsin-like activity and decreased degradation of both casein and casein modified by 4-hydroxynonenal. This loss in function could not be explained by differences in subunit composition, content of PA28 and PA700, or reversible modification of cysteine residues. To begin investigating the molecular basis for the age-associated decrement in proteasome function, we modified the cysteine residues in proteasome from young rats with the sulfhydryl reactive chemical N-ethylmaleimide. We observed inhibition of the chymotrypsin-like activity and decreased degradation of casein that was comparable to that seen in aged retinas. Thus, chemical modification of cysteine provides an in vitro method that partially recapitulates aging proteasome. Further studies are required to confirm irreversible modification of functionally significant cysteine as a potential mechanism behind the age-related loss in proteasome function.

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Section: Degradation Of Protein Substratesmentioning

confidence: 86%

Section: Content Of Regulatory Complexesmentioning

confidence: 99%

Age-dependent inhibition of proteasome chymotrypsin-like activity in the retina

Kapphahn

Bigelow

Ferrington

2007

Experimental Eye Research

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“…These agents caused an increase in proteasome activity, as determined by means of the fluorescent substrate Nsuccinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin 21 ( Figure 1). The RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximied (CHX), antioxidants such as N-acetylcysteine (NAC) or catalase, or the glucocorticoid receptor antagonist RU38486 (which does not interfere with etoposide-induced cell death) prevent both the signs of nuclear apoptosis ( Figure 1B) and proteasome activation ( Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

Proteasome activation as a critical event of thymocyte apoptosis

et al. 2000

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Caspase activation may occur in a direct fashion as a result of CD95 death receptor crosslinking (exogenous pathway) or may be triggered indirectly, via a Bcl-2 inhibitable mitochondrial permeabilization event (endogenous pathway). Thymocyte apoptosis is generally accompanied by proteasome activation. If death is induced by DNA damage, inactivation of p53, overexpression of a Bcl-2 transgene, inhibition of protein synthesis, and antioxidants (N-acetylcyteine, catalase) prevent proteasome activation. Glucocorticoid-induced proteasome activation follows a similar pattern of inhibition except for p53. Caspase inhibition fails to affect proteasome activation induced by topoisomerase inhibition or glucocorticoid receptor ligation. In contrast, caspase activation (but not p53 knockout or Bcl-2 overexpression) does interfere with proteasome activation induced by CD95. Specific inhibition of proteasomes with lactacystin or MG123 blocks caspase activation at a pre-mitochondrial level if thymocyte apoptosis is induced by DNA damage or glucocorticoids. In strict contrast, proteasome inhibition has no inhibitory effect on the mitochondrial and nuclear phases of apoptosis induced via CD95. Thus, proteasome activation is a critical event of thymocyte apoptosis stimulated via the endogenous pathway yet dispensable for CD95-triggered death. Cell Death and Differentiation (2000) 7, 368 ± 373.

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“…Substrate affinities become lower when purified proteasome particles are analyzed. For example, purified rat liver 20S proteasome has a K M value of 0.06 mmol L −1 for the same Chy-like substrate as used in this study (Reidlinger et al 1997). Various groups have investigated kinetic parameters of the proteasome (Djaballah and Rivett 1992;Cardozo et al 1995;Kisselev et al 2002).…”

Section: Discussionmentioning

confidence: 99%

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

Götze

Saborowski

Martínez‐Cruz

et al. 2017

Cell Stress and Chaperones

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Crustaceans are intensively farmed in aquaculture facilities where they are vulnerable to parasites, bacteria, or viruses, often severely compromising the rearing success. The ubiquitin-proteasome system (UPS) is crucial for the maintenance of cellular integrity. Analogous to higher vertebrates, the UPS of crustaceans may also play an important role in stress resistance and pathogen defense. We studied the general properties of the proteasome system in the hemocytes of the whiteleg shrimp, Penaeus vannamei, and the European brown shrimp Crangon crangon. The 20S proteasome was the predominant proteasome population in the hemocytes of both species. The specific activities of the trypsin-like (Try-like), chymotrypsinlike (Chy-like), and caspase-like (Cas-like) enzymes of the shrimp proteasome differed between species. P. vannamei exhibited a higher ratio of Try-like to Chy-like activities and Caslike to Chy-like activities than C. crangon. Notably, the Chy-like activity of P. vannamei showed substrate or product inhibition at concentrations of more than 25 mmol L −1 . The K M values ranged from 0.072 mmol L −1 for the Try-like activity of P. vannamei to 0.309 mmol L −1 for the Cas-like activity of C. crangon. Inhibition of the proteasome of P. vannamei by proteasome inhibitors was stronger than in C. crangon. The pH profiles were similar in both species. The Try-like, Chy-like, and Cas-like sites showed the highest activities between pH 7.5 and 8.5. The proteasomes of both species were sensitive against repeated freezing and thawing losing~80-90% of activity. This study forms the basis for future investigations on the shrimp response against infectious diseases, and the role of the UPS therein.

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Catalytic Properties of 26 S and 20 S Proteasomes and Radiolabeling of MB1, LMP7, and C7 Subunits Associated with Trypsin-like and Chymotrypsin-like Activities

Cited by 61 publications

References 54 publications

Age-dependent inhibition of proteasome chymotrypsin-like activity in the retina

Age-dependent inhibition of proteasome chymotrypsin-like activity in the retina

Proteasome activation as a critical event of thymocyte apoptosis

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

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