RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor

Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors

RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor

Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors.Peer reviewe

Structural characterization of TIR domains interaction

Receptory Toll-podobne (TLR) są odpowiedzialne za aktywację odpowiedzi wrodzonej układu immunologicznego po rozpoznaniu molekularnych wzorców patogenności. Ścieżka transdukcji sygnału receptorów TLR wymaga związania do receptora białek adaptorowych, do których zalicza się MyD88 (ang. myeloid differentiation primary response gene 88) oraz MAL (ang. MyD88 adapter-like protein). Białko MyD88 odgrywa ważną rolę w różnych procesach fizjologicznych oraz chorobotwórczych takich jak indukcja stanu zapalnego oraz proces nowotworzenia. Z kolei mutacje białka MAL kojarzone są ze zwiększoną podatnością lub opornością na niektóre choroby, takie jak bakteriemia, gruźlica czy malaria. Dlatego ważne jest poznanie molekularnych podstaw działania tych białek. Każdy TLR oraz wszystkie białka adaptorowe zawierają domenę TIR (ang. Toll/IL-1 receptor), która pełni kluczową rolę w ich interakcji. Dotychczas przedstawiono dwa możliwe modele oddziaływania domen TIR białek: MAL, MyD88 oraz TLR4. Jednak do tej pory molekularny mechanizm oddziaływania TIR:TIR pozostaje niewyjaśniony. W prezentowanej pracy wykorzystując metody biologii molekularnej uzyskano konstrukt domeny TIR białka MyD88 z metką GST, a następnie wykonano optymalizację warunków ekspresji białka. Opracowano również trzyetapowy proces oczyszczania domeny MyD88-TIR z wykorzystaniem chromatografii powinowactwa na złożu Glutathione Sepharose 4 FF, sączenia molekularnego oraz chromatografii jonowymiennej. Dzięki uprzejmości dr Eugene Valkov uzyskano konstrukt domeny TIR białka MAL z metką histydynową i opracowano protokół oczyszczania białka za pomocą chromatografii powinowactwa na złożu niklowym oraz chromatografii jonowymiennej. Wykorzystując białko MyD88-TIR znakowane izotopem azotu 15N przeprowadzono pomiary jedno- oraz dwuwymiarowego widma NMR, które potwierdziły, że białko jest poprawnie sfałdowane i nie ulega agregacji. Metodą analitycznego sączenia molekularnego pokazano, że domeny TIR białek MAL oraz MyD88 przyjmują w roztworze formę monomeru. W celu sprawdzenia funkcjonalności otrzymanych domen TIR przeprowadzono eksperyment pull-down, który potwierdził oddziaływanie obu domen. Ponadto wykorzystując metodę sączenia molekularnego zaobserwowano prawdopodobne formowanie się kompleksu domeny TIR białka MAL oraz MyD88. W celu sprawdzenia czy otrzymany kompleks jest homo- czy heterodimerem przeprowadzono rozdział elektroforetyczny, jednak wyniki te nie są klarowne i wymagają dalszej analizy.Toll-like receptors (TLRs) play central role in the innate immune response and inflammation by recognizing pathogen-associated molecular patterns. A TLR signalling depends on recruitment and association of adaptor proteins including MyD88 (myeloid differentiation primary response gene 88) and Mal (MyD88 adapter-like protein, also known as TIRAP). MyD88 takes part in various physiological and phatogenic processes such as inflammation, host defence and carcinogenesis, whereas mutations in MAL are associated with higher susceptibility or resistance to infectious diseases including bacteraemia, tuberculosis and malaria. Thus, it is important to elucidate the molecular mechanism of interaction of those adaptor proteins. All TLRs and adaptor proteins contain structurally conserved Toll/interleukin-1 receptor (TIR) domain, which is crucial in their interaction. Up to date two possible models of MyD88, Mal and TLR4 interactions have been proposed. Despite numerous reports on TIR domains, the molecular mechanism of TIR:TIR domain interactions remains an unresolved issue. In this study construct of MyD88 TIR domain with GST tag was obtained using molecular cloning methods and the conditions for protein expression were optimised. Three-step purification protocol with affinity, size-exclusion and ion-exchange chromatography has been established for MyD88 TIR domain. Construct of MAL TIR domain with histidine tag has been kindly provided by dr Eugene Valkov. Purification procedure of MAL-TIR using affinity and ion-exchange chromatography has been developed. Using NMR experiments it has been shown that MyD88 TIR domain is properly folded and does not undergo aggregation. TIR domains of MAL and MyD88 were also shown to exist as a monomers in solution state on the basis of size-exclusion chromatography. Pull-down analysis confirmed that recombinant TIR domains from MyD88 and MAL are fully functional and interact with each other. Size-exclusion chromatography demonstrated probable formation of complex of MAL and MyD88 TIR domains. To examine whether obtained complexes are homo- or heterodimers SDS-PAGE analysis was performed. However, obtained results are not clear and require further investigation

A stapled peptide mimetic of the CtIP tetramerization motif interferes with double-strand break repair and replication fork protection

Cancer cells display high levels of DNA damage and replication stress, vulnerabilities that could be exploited by drugs targeting DNA repair proteins. Human CtIP promotes homology-mediated repair of DNA double-strand breaks (DSBs) and protects stalled replication forks from nucleolytic degradation, thus representing an attractive candidate for targeted cancer therapy. Here, we establish a peptide mimetic of the CtIP tetramerization motif that inhibits CtIP activity. The hydrocarbon-stapled peptide encompassing amino acid residues 18 to 28 of CtIP (SP$^{18-28}$) stably binds to CtIP tetramers in vitro and facilitates their aggregation into higher-order structures. Efficient intracellular uptake of SP$^{18-28}$ abrogates CtIP localization to damaged chromatin, impairs DSB repair, and triggers extensive fork degradation. Moreover, prolonged SP$^{18-28}$ treatment causes hypersensitivity to DNA-damaging agents and selectively reduces the viability of BRCA1-mutated cancer cell lines. Together, our data provide a basis for the future development of CtIP-targeting compounds with the potential to treat patients with cancer

Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1

SummaryTargeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has recently provided breakthrough progress in the treatment of melanoma, non-small cell lung cancer, and other types of cancer. Small-molecule drugs interfering with this pathway are highly awaited, but their development is hindered by insufficient structural information. This study reveals the molecular details of the human PD-1/PD-L1 interaction based on an X-ray structure of the complex. First, it is shown that the ligand binding to human PD-1 is associated with significant plasticity within the receptor. Second, a detailed molecular map of the interaction surface is provided, allowing definition of the regions within both interacting partners that may likely be targeted by small molecules

CtIP-mediated fork protection synergizes with BRCA1 to suppress genomic instability upon DNA replication stress

Protecting stalled DNA replication forks from degradation by promiscuous nucleases is essential to prevent genomic instability, a major driving force of tumorigenesis. Several proteins commonly associated with the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) have been implicated in the stabilization of stalled forks. Human CtIP, in conjunction with the MRE11 nuclease complex, plays an important role in HR by promoting DSB resection. Here, we report an unanticipated function for CtIP in protecting reversed forks from degradation. Unlike BRCA proteins, which defend nascent DNA strands from nucleolytic attack by MRE11, we find that CtIP protects perturbed forks from erroneous over-resection by DNA2. Finally, we uncover functionally synergistic effects between CtIP and BRCA1 in mitigating replication-stress-induced genomic instability. Collectively, our findings reveal a DSB-resection- and MRE11-independent role for CtIP in preserving fork integrity that contributes to the survival of BRCA1-deficient cells

RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor

Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors. Keywords: BARD1; BRCA1; CtIP; DNA damage; replication stress; synthetic lethalit

H2AX promotes replication fork degradation and chemosensitivity in BRCA-deficient tumours.

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair