Cross-link between the ubiquitin-proteasome system (UPS) and autophagy in the regulation of mitophagy

Autophagy and the ubiquitin–proteasome system (UPS) are the two major intracellular protein quality control and recycling mechanisms that are responsible for cellular homeostasis in eukaryotes. Ubiquitylation is utilized as a degradation signal for both systems, however, the two system differ in terms of their mode of actions. The UPS is responsible for the degradation of short-lived proteins and soluble unfolded/misfolded proteins whereas autophagy eliminates rather long-lived proteins, insoluble protein aggregates and even whole organelles (e.g., mitochondria, peroxisomes) and pathogenic invaders (e.g., bacteria). In addition to an indirect connection between the two systems through ubiquitylated proteins, recent data indicate the presence of functional connections and reciprocal regulation mechanisms between these degradation pathways. In this thesis work, we have characterized and analyzed novel and direct links between the UPS and autophagy. Autophagy of mitochondria was chosen as a model to study the interaction and crosstalk between autophagy and the UPS. Functional consequences of these findings will be presented and discussed in detail

Crosstalk between mammalian autophagy and the ubiquitin-proteasome system

Autophagy and the ubiquitin-proteasome system (UPS) are the two major intracellular quality control and recycling mechanisms that are responsible for cellular homeostasis in eukaryotes. Ubiquitylation is utilized as a degradation signal by both systems, yet, different mechanisms are in play. The UPS is responsible for the degradation of short-lived proteins and soluble misfolded proteins whereas autophagy eliminates long-lived proteins, insoluble protein aggregates and even whole organelles (e.g., mitochondria, peroxisomes) and intracellular parasites (e.g., bacteria). Both the UPS and selective autophagy recognize their targets through their ubiquitin tags. In addition to an indirect connection between the two systems through ubiquitylated proteins, recent data indicate the presence of connections and reciprocal regulation mechanisms between these degradation pathways. In this review, we summarize these direct and indirect interactions and crosstalks between autophagy and the UPS, and their implications for cellular stress responses and homeostasis

Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy

Autophagy is an evolutionarily conserved eukaryotic cellular mechanism through which cytosolic fragments, misfolded/aggregated proteins and organelles are degraded and recycled. Priming of mitochondria through ubiquitylation is required for the clearance the organelle by autophagy (mitophagy). Familial Parkinson’s Disease-related proteins, including the E3-ligase PARK2 (PARKIN) and the serine/threonine kinase PARK6 (PINK1) control these ubiquitylation reactions and contribute to the regulation of mitophagy. Here we describe, novel protein complexes containing autophagy protein ATG5 and ubiquitin-proteasome system (UPS) components. We discovered that ATG5 interacts with PSMA7 and PARK2 upon mitochondrial stress. Results suggest that all three proteins translocate mitochondria and involve in protein complexes containing autophagy, UPS and mitophagy proteins. Interestingly, PARK2 and ATG5 recruitment onto mitochondria requires proteasome components PSMA7 and PSMB5. Strikingly, we discovered that subunit of 20 S proteasome, PSMA7, is required for the progression of PARK2-PARK6-mediated mitophagy and the proteasome activity following mitochondrial stress. Our results demonstrate direct, dynamic and functional interactions between autophagy and UPS components that contribute to the regulation of mitophagy

Identification of KLHDC2 as an efficient proximity-induced degrader of K-RAS, STK33, β-catenin, and FoxP3

Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, β-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells

IBMPFD disease-causing mutant VCP/p97 proteins are targets of autophagic-lysosomal degradation

The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies

Autophagy as a molecular target for cancer treatment

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed

Autophagy as a molecular target for cancer treatment

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sus-tainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions suchas tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful.Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed

A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress

The maintenance of cellular homeostasis in living organisms requires a balance between anabolic and catabolic reactions. Macroautophagy (autophagy herein) is determined as one of the major catabolic reactions. Autophagy is an evolutionarily conserved stress response pathway that is activated by various insults including DNA damage. All sorts of damage to DNA potentially cause loss of genetic information and trigger genomic instability. Most of these lesions are repaired by the activation of DNA damage response following DNA repair mechanisms. Here we describe, a novel protein complex containing the autophagy protein ATG5 and the non-homologous end-joining repair system proteins. We discovered for the first time that ATG5 interacted with both Ku80 (XRCC5) and Ku70 (XRCC6). This novel interaction is facilitated mainly via Ku70. Our results suggest that this interaction is dynamic and enhanced upon genotoxic stresses. Strikingly, we identified that ATG5-Ku70 interaction is necessary for DNA repair and effective recovery from genotoxic stress. Therefore, our results are demonstrating a novel, direct, dynamic, and functional interaction between ATG5 and Ku70 proteins that plays a crucial role in DNA repair under genotoxic stress conditions

P137L mutant VCP/p97, but not the WT VCP/p97 or R155C and R191Q mutants, was a target of starvation-induced autophagy.

<p>(A) HEK293T cells were transfected with empty vector (CNT), MYC-tagged wild type (WT) or mutant VCP/p97 (P137L) and then starved for indicated time points. (B and C) HEK293T cells and (D) U2OS cells expressing empty vector (CNT), wild type (WT) or mutant (P137L) VCP/p97 were starved for 40 min in the absence or presence of lysosomal inhibitors E64d/Pepstatin A (E64D/PEPA) or chloroquine (CQ). (E) HEK293T cells and (F) U2OS cells expressing other VCP/p97 mutants (R155C) and (R191Q) were starved for 40 min in the absence or presence of lysosomal inhibitor chloroquine (CQ). (G) U2OS cells expressing wild type (WT) or mutant VCP/p97 (P137L) and (R155C) were starved for 40 min in the absence or presence of lysosomal inhibitor chloroquine (CQ). (H) U2OS cells were treated with carrier (DMSO) or Torin-1 (TRN) and western blot analysis was performed by using LC3 antibody. (I) Cells were transfected with wild type (WT) and mutant (P137L or R155C) VCP/p97 and treated with TRN in the presence or absence of CQ. ACTIN was used as loading control. Image J software was used for the quantification of band intensities. Western blot analysis was performed by using MYC and ACTIN antibodies. ACTIN was used as loading control. Image J software was used for the quantification of band intensities.</p

VCP/p97 mutant P137L induced autophagy.

<p>(A) Primary structure of VCP/p97 protein and IBMPFD-related mutations. Major protein domains were shown. Bold, P137 mutant; underlined, R155, R191 and A232 mutants. (B) HEK293T cells were transfected with empty vector (CNT), MYC-tagged wild type (WT) or mutant VCP/p97 (P137L) and western blot analysis was performed using MYC, LC3 and ACTIN antibodies. ACTIN was used as loading control. LC3-I and LC3-II band intensity was quantified on scans of immunoblot films using Image J software. LC3-II/LC3-I ratios were determined after subtraction of non-band containing area intensities, and normalized to CNT condition. (C and D) WT or P137L mutant VCP/p97 were co-transfected with GFP-tagged LC3 in HEK293T cells and dot formation was visualized (C) and quantified (D). Data were shown as mean ± SD of independent experiments (n = 3) *** p<0.01.</p