RESUMO
Agents that raise cyclic guanosine monophosphate (cGMP) by activating protein kinase G increase 26S proteasome activities, protein ubiquitination and degradation of misfolded proteins. Therefore, they may be useful in treating neurodegenerative and other diseases caused by an accumulation of misfolded proteins. Mutations in myelin protein zero (MPZ) cause the peripheral neuropathy Charcot-Marie-Tooth type 1B (CMT1B). In peripheral nerves of a mouse model of CMT1B, where the mutant MPZS63del is expressed, proteasome activities are reduced, mutant MPZS63del and polyubiquitinated proteins accumulate and the unfolded protein response (p-eif2α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZS63del, but not of wild-type MPZ. Treating S63del mice with the phosphodiesterase 5 inhibitor, sildenafil-to raise cGMP-increased proteasome activity in sciatic nerves and reduced the levels of polyubiquitinated proteins, the proteasome reporter ubG76V-GFP and p-elF2α. Furthermore, sildenafil treatment reduced the number of amyelinated axons, and increased myelin thickness and nerve conduction velocity in sciatic nerves. Thus, agents that raise cGMP, including those widely used in medicine, may be useful therapies for CMT1B and other proteotoxic diseases.
Assuntos
Doença de Charcot-Marie-Tooth , Complexo de Endopeptidases do Proteassoma , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Células HEK293 , Humanos , Camundongos , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Nervo Isquiático/metabolismoRESUMO
Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Animais , Animais Geneticamente Modificados , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Fosforilação , Tauopatias , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Peixe-Zebra , Proteínas tau/metabolismoRESUMO
Pharmacological agents that raise cAMP and activate protein kinase A (PKA) stimulate 26S proteasome activity, phosphorylation of subunit Rpn6, and intracellular degradation of misfolded proteins. We investigated whether a similar proteasome activation occurs in response to hormones and under various physiological conditions that raise cAMP. Treatment of mouse hepatocytes with glucagon, epinephrine, or forskolin stimulated Rpn6 phosphorylation and the 26S proteasomes' capacity to degrade ubiquitinated proteins and peptides. These agents promoted the selective degradation of short-lived proteins, which are misfolded and regulatory proteins, but not the bulk of cell proteins or lysosomal proteolysis. Proteasome activities and Rpn6 phosphorylation increased similarly in working hearts upon epinephrine treatment, in skeletal muscles of exercising humans, and in electrically stimulated rat muscles. In WT mouse kidney cells, but not in cells lacking PKA, treatment with antidiuretic hormone (vasopressin) stimulated within 5-minutes proteasomal activity, Rpn6 phosphorylation, and the selective degradation of short-lived cell proteins. In livers and muscles of mice fasted for 12-48 hours cAMP levels, Rpn6 phosphorylation, and proteasomal activities increased without any change in proteasomal content. Thus, in vivo cAMP-PKA-mediated proteasome activation is a common cellular response to diverse endocrine stimuli and rapidly enhances the capacity of target tissues to degrade regulatory and misfolded proteins (e.g., proteins damaged upon exercise). The increased destruction of preexistent regulatory proteins may help cells adapt their protein composition to new physiological conditions.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Epinefrina/farmacologia , Glucagon/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Rim , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Deficiências na Proteostase/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Ubiquitinadas/metabolismoRESUMO
In several neurodegenerative diseases in which misfolded proteins accumulate there is impairment of the ubiquitin proteasome system (UPS). We tested if a similar disruption of proteostasis occurs in hereditary peripheral neuropathies. In sciatic nerves from mouse models of two human neuropathies, Myelin Protein Zero mutation (S63del) and increased copy number (P0 overexpression), polyubiquitinated proteins accumulated, and the overall rates of protein degradation were decreased. 26S proteasomes affinity-purified from sciatic nerves of S63del mice were defective in degradation of peptides and a ubiquitinated protein, unlike proteasomes from P0 overexpression, which appeared normal. Nevertheless, cellular levels of 26S proteasomes were increased in both, through the proteolytic-activation of the transcription factor Nrf1, as occurs in response to proteasome inhibitors. In S63del, increased amounts of the deubiquitinating enzymes USP14, UCH37, and USP5 were associated with proteasomes, the first time this has been reported in a human disease model. Inhibitors of USP14 increased the rate of protein degradation in S63del sciatic nerves and unexpectedly increased the phosphorylation of eIF2α by Perk. Thus, proteasome content, composition and activity are altered in these diseases and USP14 inhibitors have therapeutic potential in S63del neuropathy.
Assuntos
Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteólise , Nervo Isquiático/metabolismo , Animais , Feminino , Neuropatia Hereditária Motora e Sensorial/patologia , Masculino , Camundongos , Camundongos Transgênicos , Nervo Isquiático/patologia , Ubiquitina Tiolesterase/biossíntese , Ubiquitina Tiolesterase/genéticaRESUMO
The ubiquitin proteasome system degrades the great majority of proteins in mammalian cells. Countless studies have described how ubiquitination promotes the selective degradation of different cell proteins. However, there is a small but the growing literature that protein half-lives can also be regulated by post-translational modifications of the 26S proteasome. The present study reviews the ability of several kinases to alter proteasome function through subunit phosphorylation. For example, PKA (protein kinase A) and DYRK2 (dual-specificity tyrosine-regulated kinase 2) stimulate the proteasome's ability to degrade ubiquitinated proteins, peptides, and adenosine triphosphate, while one kinase, ASK1 (apoptosis signal-regulating kinase 1), inhibits proteasome function during apoptosis. Proteasome phosphorylation is likely to be important in regulating protein degradation because it occurs downstream from many hormones and neurotransmitters, in conditions that raise cyclic adenosine monophosphate or cyclic guanosine monophosphate levels, after calcium influx following synaptic depolarization, and during phases of the cell cycle. Beyond its physiological importance, pharmacological manipulation of proteasome phosphorylation has the potential to combat various diseases. Inhibitors of phosphodiesterases by activating PKA or PKG (protein kinase G) can stimulate proteasomal degradation of misfolded proteins that cause neurodegenerative or myocardial diseases and even reduce the associated pathology in mouse models. These observations are promising since in many proteotoxic diseases, aggregation-prone proteins impair proteasome function, and disrupt protein homeostasis. Conversely, preventing subunit phosphorylation by DYRK2 slows cell cycle progression and tumor growth. However, further research is essential to determine how phosphorylation of different subunits by these (or other) kinases alters the properties of this complex molecular machine and thus influence protein degradation rates.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Animais , Apoptose , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Humanos , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , ProteóliseRESUMO
[This corrects the article DOI: 10.3389/fnmol.2019.00177.].
RESUMO
Rates of degradation by the ubiquitin proteasome system depend not only on rates of ubiquitination, but also on the level of proteasome activity which can be regulated through phosphorylation of proteasome subunits. Many protein kinases have been proposed to influence proteasomal activity. However, for only two is there strong evidence that phosphorylation of a specific 26S subunit enhances the proteasome's capacity to degrade ubiquitinated proteins and promotes protein breakdown in cells: (1) protein kinase A (PKA), which after a rise in cAMP phosphorylates the 19S subunit Rpn6, and (2) dual tyrosine receptor kinase 2 (DYRK2), which during S through M phases of the cell cycle phosphorylates the 19S ATPase subunit Rpt3. In this chapter, we review and discuss the different methods used to assess the impact of phosphorylation by these two kinases on proteasomal activity and intracellular protein degradation. In addition, we present one method to determine if phosphorylation is responsible for an observed increase in proteasomal activity and another to evaluate by Phos-tag gel electrophoresis whether a specific proteasome subunit is modified by phosphorylation. The methods reviewed and presented here should be useful in clarifying the roles of other kinases and other posttranslational modifications of proteasome subunits.