Live cell transcription-coupled nucleotide excision repair dynamics revisited

Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags.</p

N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death

DDA1, a novel factor in transcription-coupled repair, modulates CRL4CSA dynamics at DNA damage-stalled RNA polymerase II

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.<br/

MCPIP1 influence on the activation of NF-κB and MAP kinase signal transduction pathways in HepG2 cells stimulated with interleukin-1β.

Acute phase reaction represents a complex reaction of an organism to the local or systemic disorder of homeostasis, caused by infections, thermal and mechanic stress, etc. Proinflammatory cytokines (for example IL-1β) play an essential role in the regulation of acute phase reaction. Interaction between interleukin 1β and receptor IL-1RI leads to activation of intracellular processes which are involved in the regulation of NF-κB and MAPK pathways. The final stage consists of activation and translocation of transcription factors such as AP-1 and NF-κB to the nucleus. Finally, expression of genes, that are crucial for propagation of acute phase reaction, occurs. Nowadays, proteins involved in negative regulation of NF-κB pathway, are intensively studied. One of those proteins is MCPIP1. The protein was identified as a factor induced by chemokine MCP-1 in human peripheral blood monocytes. Mechanism by which MCPIP1 acts on NF-κB signaling pathway, is currently unknown.The interaction between MCPIP1 and proteins of NF-κB pathway (TANK, TRAF2, TRAF6) was examined both in unstimulated and stimulated with IL-1β HepG2 cells. The influence of MCPIP1 on amount of TANK, TRAF2 and TRAF6 during IL-1β stimulation was determined. Additionally, the influence of MCPIP1 on degradation of IκBα and translocation of p65 (subunit of NF-κB complex) to the nucleus after IL-1β treatment, was studied. The MCPIP1 cellular localization after IL-1β stimulation was examined. Furthermore, the influence of MCPIP1 on MAPK phosphorylation status (ERK, JNK and p38) after IL-1β treatment was analyzed.Results are consistent with those presented by Fu et al. [87]. Additionally, experiments where amount of TANK, TRAF2 and TRAF6 during IL-1β stimulation and cellular localization of MCPIP1 were determined, give new insight in MCPIP1 activity. Obtained results show direction of future research, which finally may lead to disclosure a detailed mechanism of MCPIP1 action on NF-κB pathway.Reakcja ostrej fazy to złożona, systemowa odpowiedź organizmu na lokalne lub ogólnoustrojowe zaburzenia homeostazy, spowodowane m.in. infekcjami różnego typu, stresem termicznym lub mechanicznym. Istotną rolę w regulacji reakcji ostrej fazy pełnią cytokiny prozapalne m.in. IL-1β. Przyłączenie cząsteczki interleukiny 1 β do receptora typu I, powoduje aktywację wewnątrzkomórkowych procesów zaangażowanych w regulację szlaku NF-κB oraz kaskady kinaz MAP. Etapem końcowym jest aktywacja oraz translokacja do jądra komórkowego czynników transkrypcyjnych m.in. AP-1 i NF-κB, które odpowiadają za ekspresję genów kluczowych dla propagacji reakcji ostrej fazy. Obecnie intensywnie badane są białka zaangażowane w negatywną regulację szlaku NF-κB. Jednym z nich jest MCPIP1, zidentyfikowany jako czynnik odpowiedzi ludzkich monocytów krwi obwodowej na stymulację MCP1. Mechanizm poprzez który MCPIP1 hamuje aktywację kompleksu NF-κB nie jest znany. W ramach pracy magisterskiej zbadano oddziaływanie pomiędzy MCPIP1, a białkami szlaku NF-κB: TANK, TRAF2 i TRAF6, zarówno w warunkach braku stymulacji jak i stymulacji IL-1β. Określono wpływ MCPIP1 na poziom wymienionych białek po stymulacji IL-1β. Ponadto, sprawdzono wpływ MCPIP1 na degradację inhibitora IκBα oraz na translokację p65 (podjednostki kompleksu NF-κB) do jądra komórkowego po stymulacji IL-1β. Dodatkowo, zbadano wpływ stymulacji IL-1β na lokalizację komórkową MCPIP1 w komórkach linii HepG2. Sprawdzono też wpływ MCPIP1 na fosforylację kinaz ERK, JNK i p38 po stymulacji IL-1β. Uzyskane wyniki potwierdzają rezultaty Fu i wsp. [87], a także wprowadzają nowe informacje dotyczące wpływu MCPIP1 na ilość TANK, TRAF2 i TRAF6 oraz lokalizację MCPIP1 po stymulacji komórek IL-1β. Ponadto, rezultaty wykonanych doświadczeń wskazują kierunek dalszych badań, mających na celu szczegółowe przedstawienie mechanizmu działania MCPIP1 na szlak NF-κB

Poland and Poles in Norwegian press

Rozróżnienie znaczenia terminów: obraz, stereotyp i wyobrażenia społeczne. Przedstawienie historii relacji polsko-norweskich od średniowiecza do czasów współczesnych. Obraz Polski i Polaków na podstawie analizy artykułów zamieszczonych w internetowej prasie norweskiej.Differentiating the meaning of terms: image, stereotype, and social representations. Presentation of the history of Polish-Norwegian relations from the Middle Ages to the present times. Image of Poland and Poles based on the analysis of the articles published in the Norwegian press

Resolving the complexity of ubiquitin networks

Ubiquitination regulates nearly all cellular processes by coordinated activity of ubiquitin writers (E1, E2, and E3 enzymes), erasers (deubiquitinating enzymes) and readers (proteins that recognize ubiquitinated proteins by their ubiquitin-binding domains). By differentially modifying cellular proteome and by recognizing these ubiquitin modifications, ubiquitination machinery tightly regulates execution of specific cellular events in space and time. Dynamic and complex ubiquitin architecture, ranging from monoubiquitination, multiple monoubiquitination, eight different modes of homotypic and numerous types of heterogeneous polyubiquitin linkages, enables highly dynamic and complex regulation of cellular processes. We discuss available tools and approaches to study ubiquitin networks, including methods for the identification and quantification of ubiquitin-modified substrates, as well as approaches to quantify the length, abundance, linkage type and architecture of different ubiquitin chains. Furthermore, we also summarize the available approaches for the discovery of novel ubiquitin readers and ubiquitin-binding domains, as well as approaches to monitor and visualize activity of ubiquitin conjugation and deconjugation machineries. We also discuss benefits, drawbacks and limitations of available techniques, as well as what is still needed for detailed spatiotemporal dissection of cellular ubiquitination network

N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.</p

DDA1, a novel factor in transcription-coupled repair, modulates CRL4CSA dynamics at DNA damage-stalled RNA polymerase II

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.<br/