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Title: Role of calpain in USP1 stability regulation and genome integrity maintenance
Other Titles: Ruolo di calpaina nella regolazione della stabilità di USP1 e nel mantenimento dell'integrità genomica
Authors: Cataldo, Francesca
Supervisore/Tutore: Schneider, Claudio
Cosupervisore: Demarchi, Francesca
Issue Date: 24-Apr-2012
Publisher: Università degli studi di Trieste
The calpains are a family of intracellular cysteine proteases, among which the best studied isoforms, micro- (CAPN1) and milli-calpain (CAPN2), are heterodimers consisting of a catalytic subunit and a common regulatory subunit, CAPNS1, required for function. Calpain is involved in many processes important for cancer biology, such as autophagy, indeed in calpain-depleted cells autophagy is impaired, with a subsequent increase in apoptosis sensitivity. Calpain is also important in all the stages of the stress response.
A proteomic approach was employed for the identification of novel CAPNS1 interacting proteins. Proteins immunoprecipitating with endogenous CAPNS1 in HT1080 cell lysates were analyzed by Mass Spectrometry. We identified novel partners among which the deubiquitinating enzyme USP1, a key regulator of the DNA damage response and genome integrity maintenance via its specific action on FANCD2, involved in DNA repair and protection from chromosome instability, and PCNA, involved in the regulation of translesion DNA synthesis (TLS), that bypasses DNA lesions with low stringency basepairing requirements.
We performed co-IP assays in lysates of 293T cells and confirmed that the interaction was specific. Furhermore, we observed that calpain is able to bind a USP1 C-terminal deleted mutant, suggesting that USP1 first 523 aminoacids were sufficient for the binding.
To understand what is the effect exerted by calpain upon USP1, we depleted calpain activity in a series of cell lines, and followed the fate of endogenous USP1. We transfected CAPNS1 specific siRNAs, or treated cells with a specific inhibitor of calpain, and we observed a strong decrease in USP1 protein levels. This effect should be at a post-transcriptional level, since any significant change in USP1 mRNA levels is detected. We also obtained the same result by transfecting a siRNA specific for CAPN1, the gene encoding for the catalytic subunit micro-calpain.
Moreover, we studied the role of calpain in the PCNA-mediated switch between high fidelity replication and TLS upon UV irradiation. In mouse embryonic fibroblasts knockout for CAPNS1, USP1 downregulation is coupled to an increase in PCNA monoubiquitination. Moreover, CAPNS1-depleted U2OS cells showed an increase in the percentage of nuclei containing PCNA-induced foci upon UV irradiation.
Since we demonstrated that calpain can modulate an important regulator of DNA damage response such as USP1, we investigated if calpain could have a role in genome integrity maintenance. CAPNS1 depleted cells showed a reduced rescue in DNA repair compared to control cells, suggesting that increased levels in PCNA monoubiquitination could lead to an increased amount of errore-prone TLS.
Calpain plays an important role in autophagy, so we asked if USP1 degradation in absence of calpain activity could involve autophagic pathways. We first blocked macroautophagy by silencing ATG5, and we observed that USP1 was downregulated, suggesting that the depletion of ATG5 could lead to an increased activity of other degradation pathways. To impaire chaperone-mediated autophagy (CMA), we silenced a protein important for autophagosome formation, LAMP-2A. Also in this case we observed a decrease in USP1 protein levels, thus suggesting that USP1 is alternatively degraded by different pathways. However, we observed that USP1 is stabilized upon inhibition of lysosomal enzymes, suggesting that USP1 may be degraded in the lysosome.
To better understand the mechanism by which calpain affect USP1 stability we search for an effect of calpain upon USP1 co-factor and activator UAF1/WDR48. CAPNS1-depleted cells showed WDR48 downregulation, but WDR48 overexpression only partially rescue USP1 protein levels in this cells. Furthermore, we provided evidences that calpain regulation of p35/p25 activator of Cdk5 can affect Cdh1 phosphorylation and thus APC/Cdh1 activity, leading to a regulation of USP1 stabilization.
In conclusion, we identified USP1 as a novel interactor of calpain, and we found that calpain is important for USP1 stability, since in its absence USP1 is downregulated. The importance of this novel regulation is strengthened by the recent findings that unveiled a role of USP1 in maintenance of a mesenchymal stem cell program in osteosarcoma, and thus placing calpain in a crucial regulatory position for cancer development.
Ciclo di dottorato: XXIV Ciclo
Keywords: Calpain systemUbiquitin-Specific Peptidase 1DNA damage responseTranslesion synthesisAPC/C-Cdh1
Type: Doctoral
Language: en
Settore scientifico-disciplinare: BIO/11 BIOLOGIA MOLECOLARE
NBN: urn:nbn:it:units-4462
Appears in Collections:Scienze biologiche

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