Regulation of 26S Proteasome Activity in Pulmonary Fibrosis

Welk, Vanessa; Korfei, Martina; Keller, Ilona; Fernandez, Isis E.; Adler, Heiko; Günther, Andreas; Eickelberg, Oliver; Meiners, Silke

doi:10.1164/rccm.201412-2270oc

Cited by 39 publications

(47 citation statements)

References 55 publications

(36 reference statements)

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“…Primary Cell Culture-Primary human lung fibroblasts (phLF) were isolated from organ donor lungs and cultured as previously described (37)(38)(39). 24 h prior to treatment phLF were synchronized by incubation in starvation medium containing 1% FBS.…”

Section: Methodsmentioning

confidence: 99%

“…1, 4390843, and Negative Control No. 2, 4390847, Ambion, Life Technologies) at a final concentration of 0.5 nM for RPN6 silencing and 2 nM for PA28␥ silencing using Lipofectamine RNAiMAX (13778150, Life Technologies) as previously reported (39).…”

Section: Methodsmentioning

confidence: 99%

“…Lysates were centrifuged at maximum speed for 20 min at 4°C and subjected to electrophoresis using the XCell SureLock Mini-Cell system (EI0001, Life Technologies). 15 g of protein were run on 3-8% Tris acetate gradient gels (EA0378BOX, Life Technologies) for 3-4 h at 150 V. Chymotrypsin-like activity was monitored as described previously (39). After denaturing in solubilization buffer (2% (w/v) SDS, 66 mM Na 2 CO 3 , 1.5% ␤-mercaptoethanol) proteasome complexes were blotted under normal conditions.…”

Section: Methodsmentioning

confidence: 99%

“…of Only PA200 Alternative Proteasomes-To further assess whether formation of alternative proteasome complexes and induction of PA28␥ and PA200 expression at later time points involves ubiquitin-mediated protein degradation, we selectively inhibited 26S/30S ubiquitin-mediated protein degradation by partial knockdown of the 19S subunit RPN6 (39,47,48).…”

Section: Inhibition Of 26s/30s Proteasome Activity Induces Formationmentioning

confidence: 99%

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Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

Welk

Coux

Kleene

et al. 2016

Journal of Biological Chemistry

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The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28␣␤, PA28␥, PA200, and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28␥ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28␥ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of ␤5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA-mediated knockdown of the 19S subunit RPN6 induced recruitment of only PA200 to 20S proteasomes, whereas PA28␥ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28␥ sensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis.The proteasome, one of the main proteolytic systems of the cell, is essential for maintenance of protein homeostasis and thus of cellular functions. The proteolytic activity of this multicatalytic protease resides inside the 20S core particle, built of four stacked rings of seven ␣ and ␤ subunits with ␣ 7

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Inhibition Of 26s/30s Proteasome Activity Induces Formationmentioning

confidence: 99%

See 2 more Smart Citations

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

Welk

Coux

Kleene

et al. 2016

Journal of Biological Chemistry

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“…Changes in proteostasis lead to an increase of nonfunctional proteins, accumulation of cytotoxic proteins, and/or aggregation of misfolded proteins. In IPF lungs, evidence of altered proteostasis has been found at different levels, including protein misfolding, markers of ER stress (99)(100)(101)(102), defective autophagy, and impaired proteasome activity (103,104) (Table 4). For instance, familial pulmonary fibrosis is associated with several mutations in surfactant A and C genes (105)(106)(107)(108).…”

Section: Cellular Perturbations In the Ipf Lungmentioning

confidence: 99%

Mitochondria in the spotlight of aging and idiopathic pulmonary fibrosis

Mora¹,

Bueno²,

Rojas³

2017

Journal of Clinical Investigation

176

169

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One of the most remarkable medical accomplishments in the last century has been the increasing longevity of the human population. A decrease in birth rates, accompanied by a reduction in mortality among the elderly population, has collectively increased the percentage of the aged population, particularly in developed countries. Globally, it is estimated that the proportion of the population over the age of 60 will increase from 11% to 22% by 2050, and 28% by 2100. Currently, 18% of the US population is over 60 years of age (57 million), which is predicted to rise to 27% (107 million) in 2050 and up to 31% (149 million) by 2100 (1). The aging population has propelled an increase in the overall prevalence of chronic conditions. Several lung diseases, including acute lung injury and asthma, and some infectious diseases, such as pneumonia, increase in severity and mortality with age. Additionally, there has been a significant increase in incidence of chronic lung diseases -including chronic obstructive pulmonary disease, most forms of lung cancer, and idiopathic pulmonary fibrosis (IPF) -resulting in aging-related increases in prevalence.IPF is the most common form of idiopathic interstitial lung diseases. Its overall incidence in the US is 7 to 17 per 100,000 persons with a prevalence between 13.2 and 63 per 100,000 persons (2). A 1996 study initially demonstrated a higher incidence and prevalence of IPF in patients over 65 (3). These observations were confirmed more recently by Raghu et al. (4) and others, using both broad and narrow case-finding criteria for IPF diagnosis. These studies estimated that in the US, the prevalence of IPF increases with age, ranging from 4 per 100,000 for population aged between 18 and 34 years old to 227.2 per 100,000 among those 75 or older (4). This aging-related increase in cumulative incidence and prevalence of IPF has been corroborated by several studies that include populations in Europe and Asia (5-9). Accordingly, the median age at diagnosis of sporadic IPF ranges between 50 and 85 years old, and patients younger than 50 are more commonly associated with familial, rather than sporadic, forms of the disease (2). Mortality rates from IPF are steadily increasing worldwide, and a positive association has also been observed with advanced age (10). Examination of US government health insurance data for people aged 65 and older shows an increasing prevalence of IPF among older age groups (11). These studies confirm the growing concern that aging, and consequently age-related diseases, will drive up health care expenditures. As the elderly population in developed countries is expected to double in the next 25 years, there is an urgent need to understand the pathogenesis of IPF and develop interventions to attenuate or reverse lung fibrosis. The physiology of aging and the lungEvolutionary theories of aging include the concept of selection shadow that permits the accumulation of mutations with late deleterious effects, and the theory of antagonistic pleiotropy, which supports the sele...

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Approaches to Evaluate the Impact of a Small‐Molecule Binder to a Noncatalytic Site of the Proteasome

2021

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Proteasome activity is crucial for cell survival and proliferation. In recent years, small molecules have been discovered that can affect the catalytic activity of the proteasome. Rather than targeting the active sites of the proteasome, it might be possible to affect ubiquitin‐dependent degradation of proteins by limiting the association of the 19S regulatory particle (19S RP) with the 20S core particle (20S CP) of the proteasome. We recently described the discovery of TXS‐8, a peptoid that binds to Rpn‐6. Rpn‐6 is a proteasome‐associated protein that makes critical contacts with the 19S RP and the 20S CP. Herein, we present a general workflow to evaluate the impact of a small‐molecule binder on proteasome activity by using TXS‐8 as an example. This workflow contains three steps in which specific probes or overexpressed proteins in cells are used to determine whether the hydrolysis activity of the proteasome is affected. Although, in our case, TXS‐8 did not affect proteasome activity, our workflow is highly amenable to studying a variety of small‐molecule–proteasome subunit interactions.

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Regulation of 26S Proteasome Activity in Pulmonary Fibrosis

Abstract: We identified Rpn6-dependent 26S proteasome activation as an essential feature of myofibroblast differentiation in vitro and in vivo, and our results suggest it has an important role in IPF pathogenesis.

Cited by 39 publications

References 55 publications

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

Mitochondria in the spotlight of aging and idiopathic pulmonary fibrosis

Approaches to Evaluate the Impact of a Small‐Molecule Binder to a Noncatalytic Site of the Proteasome

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