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260


Critical role for IL-1beta in DNA damage-induced mucositis

Kanarek, Naama; Grivennikov, Sergei I; Leshets, Michael; Lasry, Audrey; Alkalay, Irit; Horwitz, Elad; Shaul, Yoav D; Stachler, Matthew; Voronov, Elena; Apte, Ron N; Pagano, Michele; Pikarsky, Eli; Karin, Michael; Ghosh, Sankar; Ben-Neriah, Yinon
beta-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-kappaB inhibitor IkappaB. To appreciate tissue-specific roles of beta-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1beta as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1beta is induced by DNA damage via an NF-kappaB-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-kappaB, with failure to express the endogenous IL-1beta receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1beta prevents epithelial tight junction dysfunction and alleviates mucositis in beta-TrCP-deficient mice. IL-1beta antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.
PMCID:3926043
PMID: 24469832
ISSN: 0027-8424
CID: 836712

The ubiquitin proteasome system - Implications for cell cycle control and the targeted treatment of cancer

Bassermann, Florian; Eichner, Ruth; Pagano, Michele
Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin-proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (anaphase promoting complex or cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/CCdc20) and establishes a stable G1 phase (APC/CCdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System DA:PIT:REV.
PMCID:3694769
PMID: 23466868
ISSN: 0006-3002
CID: 539812

Protein degradation regulates centriole disengagement in mitosis [Meeting Abstract]

Pagan, J K; Jones, M; Marzio, A; Pagano, M
During S and G2 phases of the cell cycle, the tight connection between mother and daughter centrioles, termed engagement, limits centriole duplication to once per cell cycle. Late in mitosis, parent and daughter centrioles disengage and lose their typical orthogonal orientation. This process is a prerequisite for centriole duplication in the next cell cycle, and requires both PLK1 and Separase, however, while several Separase substrates are known, no PLK1 substrates have been identified. We have discovered that disassembly of the pericentriolar material in mitosis depends on PLK1 activity and SCF-mediated proteasome activity. Furthermore, our data indicates that the elimination of the pericentriolar material is essential for centriole disengagement. We will present data that, collectively, provides a molecular mechanism by which PLK1 controls centriole disengagement, demonstrating a novel requirement for protein degradation during this crucial cellular process
EMBASE:71411531
ISSN: 1059-1524
CID: 884372

Mechanisms and function of substrate recruitment by F-box proteins

Skaar, Jeffrey R; Pagan, Julia K; Pagano, Michele
S phase kinase-associated protein 1 (SKP1)-cullin 1 (CUL1)-F-box protein (SCF) ubiquitin ligase complexes use a family of F-box proteins as substrate adaptors to mediate the degradation of a large number of regulatory proteins involved in diverse processes. The dysregulation of SCF complexes and their substrates contributes to multiple pathologies. In the 14 years since the identification and annotation of the F-box protein family, the continued identification and characterization of novel substrates has greatly expanded our knowledge of the regulation of substrate targeting and the roles of F-box proteins in biological processes. Here, we focus on the evolution of our understanding of substrate recruitment by F-box proteins, the dysregulation of substrate recruitment in disease and potential avenues for F-box protein-directed disease therapies.
PMCID:3827686
PMID: 23657496
ISSN: 1471-0072
CID: 353272

FBXL2- and PTPL1-mediated degradation of p110-free p85beta regulatory subunit controls the PI(3)K signalling cascade

Kuchay, Shafi; Duan, Shanshan; Schenkein, Emily; Peschiaroli, Angelo; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Pagano, Michele
F-box proteins are the substrate-recognition subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Purification of the F-box protein FBXL2 identified the PI(3)K regulatory subunit p85beta and tyrosine phosphatase PTPL1 as interacting proteins. FBXL2 interacts with the pool of p85beta that is free of p110 PI(3)K catalytic subunits and targets this pool for ubiquitylation and subsequent proteasomal degradation. FBXL2-mediated degradation of p85beta is dependent on the integrity of its CaaX motif. Whereas most SCF substrates require phosphorylation to interact with their F-box proteins, phosphorylation of p85beta on Tyr 655, which is adjacent to the degron, inhibits p85beta binding to FBXL2. Dephosphorylation of phospho-Tyr-655 by PTPL1 stimulates p85beta binding to and degradation through FBXL2. Finally, defects in the FBXL2-mediated degradation of p85beta inhibit the binding of p110 subunits to IRS1, attenuate the PI(3)K signalling cascade and promote autophagy. We propose that FBXL2 and PTPL1 suppress p85beta levels, preventing the inhibition of PI(3)K by an excess of free p85 that could compete with p85-p110 heterodimers for IRS1.
PMCID:3865866
PMID: 23604317
ISSN: 1465-7392
CID: 316032

SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket

Xing, Weiman; Busino, Luca; Hinds, Thomas R; Marionni, Samuel T; Saifee, Nabiha H; Bush, Matthew F; Pagano, Michele; Zheng, Ning
The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCF(FBXL3) ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3-SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination.
PMCID:3618506
PMID: 23503662
ISSN: 0028-0836
CID: 306182

FBH1 protects melanocytes from transformation and is deregulated in melanomas

Jeong, Yeon-Tae; Cermak, Lukas; Guijarro, Maria V; Hernando, Eva; Pagano, Michele
FBH1 is a member of the UvrD family of DNA helicases and plays a crucial role in the response to DNA replication stress. In particular, upon DNA replication stress, FBH1 promotes double-strand breakage and activation of the DNA-PK and ATM signaling cascades in a helicase-dependent manner. In the present manuscript, we show that FBH1 is often deleted or mutated in melanoma cells, which results in their increased survival in response to replicative stress. Accordingly, FBH1 depletion promotes UV-mediated transformation of human melanocytes. Thus, FBH1 inactivation appears to contribute to oncogenic transformation by allowing survival of cells undergoing replicative stress due to external factors such as UV irradiation.
PMCID:3646868
PMID: 23466708
ISSN: 1551-4005
CID: 287222

Role of the ubiquitin proteasome system in the heart

Pagan, Julia; Seto, Tiffany; Pagano, Michele; Cittadini, Antonio
Proper protein turnover is required for cardiac homeostasis and, accordingly, impaired proteasomal function appears to contribute to heart disease. Specific proteasomal degradation mechanisms underlying cardiovascular biology and disease have been identified, and such cellular pathways have been proposed to be targets of clinical relevance. This review summarizes the latest literature regarding the specific E3 ligases involved in heart biology, and the general ways that the proteasome regulates protein quality control in heart disease. The potential for therapeutic intervention in Ubiquitin Proteasome System function in heart disease is discussed.
PMID: 23538275
ISSN: 0009-7330
CID: 306262

Regulation of the CRL4(Cdt2) Ubiquitin Ligase and Cell-Cycle Exit by the SCF(Fbxo11) Ubiquitin Ligase

Rossi, Mario; Duan, Shanshan; Jeong, Yeon-Tae; Horn, Moritz; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Antebi, Adam; Pagano, Michele
F-box proteins and DCAF proteins are the substrate binding subunits of the Skp1-Cul1-F-box protein (SCF) and Cul4-RING protein ligase (CRL4) ubiquitin ligase complexes, respectively. Using affinity purification and mass spectrometry, we determined that the F-box protein FBXO11 interacts with CDT2, a DCAF protein that controls cell-cycle progression, and recruits CDT2 to the SCF(FBXO11)complex to promote its proteasomal degradation. In contrast to most SCF substrates, which exhibit phosphodegron-dependent binding to F-box proteins, CDK-mediated phosphorylation of Thr464 present in the CDT2 degron inhibits recognition by FBXO11. Finally, our results show that the functional interaction between FBXO11 and CDT2 is evolutionary conserved from worms to humans and plays an important role in regulating the timing of cell-cycle exit.
PMCID:3624904
PMID: 23478441
ISSN: 1097-2765
CID: 271332

USP33 regulates centrosome biogenesis via deubiquitination of the centriolar protein CP110

Li, Ji; D'Angiolella, Vincenzo; Seeley, E Scott; Kim, Sehyun; Kobayashi, Tetsuo; Fu, Wenxiang; Campos, Eric I; Pagano, Michele; Dynlacht, Brian David
Centrosome duplication is critical for cell division, and genome instability can result if duplication is not restricted to a single round per cell cycle. Centrosome duplication is controlled in part by CP110, a centriolar protein that positively regulates centriole duplication while restricting centriole elongation and ciliogenesis. Maintenance of normal CP110 levels is essential, as excessive CP110 drives centrosome over-duplication and suppresses ciliogenesis, whereas its depletion inhibits centriole amplification and leads to highly elongated centrioles and aberrant assembly of cilia in growing cells. CP110 levels are tightly controlled, partly through ubiquitination by the ubiquitin ligase complex SCF(cyclin F) during G2 and M phases of the cell cycle. Here, using human cells, we report a new mechanism for the regulation of centrosome duplication that requires USP33, a deubiquitinating enzyme that is able to regulate CP110 levels. USP33 interacts with CP110 and localizes to centrioles primarily in S and G2/M phases, the periods during which centrioles duplicate and elongate. USP33 potently and specifically deubiquitinates CP110, but not other cyclin-F substrates. USP33 activity antagonizes SCF(cyclin F)-mediated ubiquitination and promotes the generation of supernumerary centriolar foci, whereas ablation of USP33 destabilizes CP110 and thereby inhibits centrosome amplification and mitotic defects. To our knowledge, we have identified the first centriolar deubiquitinating enzyme whose expression regulates centrosome homeostasis by countering cyclin-F-mediated destruction of a key substrate. Our results point towards potential therapeutic strategies for inhibiting tumorigenesis associated with centrosome amplification.
PMCID:3815529
PMID: 23486064
ISSN: 0028-0836
CID: 242352