Svetlana V. Khoronenkova

Activity-Based Chemical Proteomics Accelerates Inhibitor Development for Deubiquitylating Enzymes

Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair.

D-Amino acid oxidase: structure, catalytic mechanism, and practical application.

D-Amino acid oxidase (DAAO) is a FAD-dependent enzyme that plays an important role in microbial metabolism, utilization of endogenous D-amino acids, regulation of the nervous system, and aging in mammals. DAAO from yeasts Rhodotorula gracilis and Trigonopsis variabilis are used to convert cephalosporin C into 7-aminocephalosporanic acid, the precursor of other semi-synthetic cephalosporins. This review summarizes the recent data on the enzyme localization, physiological role, gene cloning and expression, and the studies on the enzyme structure, stability, catalytic mechanism, and practical applications.

Ubiquitin ligase ARF‐BP1/Mule modulates base excision repair

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady‐state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF‐BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase β (Pol β), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol β and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol β and increased DNA repair. Our findings provide a novel mechanism regulating steady‐state levels of BER proteins.

ATM-Dependent Downregulation of USP7/HAUSP by PPM1G Activates p53 Response to DNA Damage

Summary The deubiquitylation enzyme USP7/HAUSP plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the DNA damage response. Despite this important role in controlling other proteins, USP7 itself has not been recognized as a target for regulation. Here, we report that USP7 regulation plays a central role in DNA damage signal transmission. We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2. Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53. After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53. Our findings provide a quantitative transmission mechanism of the DNA damage signal to coordinate a p53-dependent DNA damage response.

USP47 Is a Deubiquitylating Enzyme that Regulates Base Excision Repair by Controlling Steady-State Levels of DNA Polymerase β

DNA base excision repair (BER) is an essential cellular process required for genome stability, and misregulation of BER is linked to premature aging, increased rate of mutagenesis, and cancer. We have now identified the cytoplasmic ubiquitin-specific protease USP47 as the major enzyme involved in deubiquitylation of the key BER DNA polymerase (Pol β) and demonstrate that USP47 is required for stability of newly synthesized cytoplasmic Pol β that is used as a source for nuclear Pol β involved in DNA repair. We further show that knockdown of USP47 causes an increased level of ubiquitylated Pol β, decreased levels of Pol β, and a subsequent deficiency in BER, leading to accumulation of DNA strand breaks and decreased cell viability in response to DNA damage. Taken together, these data demonstrate an important role for USP47 in regulating DNA repair and maintaining genome integrity.

D-Amino acid oxidase: Physiological role and applications

D-Amino acids play a key role in regulation of many processes in living cells. FAD-dependent D-amino acid oxidase (DAAO) is one of the most important enzymes responsible for maintenance proper level of D-amino acids. The most interesting and important data for regulation of the nervous system, hormone secretion, and other processes by D-amino acids as well as development of different diseases under changed DAAO activity are presented. The mechanism of regulation is complex and multi-parametric because the same enzyme simultaneously influences the level of different D-amino acids, which can result in opposing effects. Use of DAAO for diagnostic and therapeutic purposes is also considered.

USP7/HAUSP stimulates repair of oxidative DNA lesions

USP7 is involved in the cellular stress response by regulating Mdm2 and p53 protein levels following severe DNA damage. In addition to this, USP7 may also play a role in chromatin remodelling by direct deubiquitylation of histones, as well as indirectly by regulating the cellular levels of E3 ubiquitin ligases involved in histone ubiquitylation. Here, we provide new evidence that USP7 modulated chromatin remodelling is important for base excision repair of oxidative lesions. We show that transient USP7 siRNA knockdown did not change the levels or activity of base excision repair enzymes, but significantly reduced chromatin DNA accessibility and consequently the rate of repair of oxidative lesions.

ATM prevents DSB formation by coordinating SSB repair and cell cycle progression

Significance DNA is chemically unstable; therefore, human cells have to repair a great number and variety of DNA lesions each day. DNA single-strand breaks (SSBs) represent one of the most dangerous lesions because, if unrepaired, they lead to genetic instability and are implicated in many human diseases. Here we report a novel mechanism that detects SSBs and controls the timing of SSB repair, thereby preventing the duplication of damaged DNA and the accumulation of mutations. Our findings shed important light on the molecular defects associated with the human disease ataxia-telangiectasia, which is caused by mutations in the atm gene. DNA single-strand breaks (SSBs) arise as a consequence of spontaneous DNA instability and are also formed as DNA repair intermediates. Their repair is critical because they otherwise terminate gene transcription and generate toxic DNA double-strand breaks (DSBs) on replication. To prevent the formation of DSBs, SSB repair must be completed before DNA replication. To accomplish this, cells should be able to detect unrepaired SSBs, and then delay cell cycle progression to allow more time for repair; however, to date there is no evidence supporting the coordination of SSB repair and replication in human cells. Here we report that ataxia-telangiectasia mutated kinase (ATM) plays a major role in restricting the replication of SSB-containing DNA and thus prevents DSB formation. We show that ATM is activated by SSBs and coordinates their repair with DNA replication. SSB-mediated ATM activation is followed by a G1 cell cycle delay that allows more time for repair and thus prevents the replication of damaged DNA and DSB accrual. These findings establish an unanticipated role for ATM in the signaling of DNA SSBs and provide important insight into the molecular defects leading to genetic instability in patients with ataxia-telangiectasia.

ATMIN is a transcriptional regulator of both lung morphogenesis and ciliogenesis

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmingpg6/gpg6, AtminH210Q/H210Q and Dynll1GT/GT, revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1GT/GT embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.