Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X.

USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual ß-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the ß-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.

Exploring Community Stakeholders' Perceptions of the Enhancing Family Well-being Project in Hong Kong: A Qualitative Study.

BACKGROUND: Community engagement is a powerful tool in bringing about positive social and community change. Community stakeholders possess critical experience and knowledge that are needed to inform the development of community-based projects. However, limited literature is available on the practical experience involved with planning and implementing community-based family programs. Even less has been published documenting efforts in Chinese communities. This paper explores community stakeholders' experiences with the enhancing family well-being project-part of a citywide project entitled the "FAMILY Project," aimed at promoting family health, happiness, and harmony in Hong Kong. METHODS: This qualitative evaluation examined the perspectives of community stakeholders. Four focus groups with social workers (n = 24) and six in-depth interviews with steering committee members were conducted from December 2012 to May 2013 in Hong Kong. Focus groups and in-depths interview were audiotaped, transcribed, and analyzed using thematic analysis techniques. RESULTS: Rich accounts were given by our respondents on various aspects of the project. Main themes and subthemes were identified and grouped into four categories (project conception, project implementation, project consolidation, and the overall impact of the project). Respondents described the practical challenges associated with the project (e.g., recruitment, balancing scientific research, and lack of resources) and identified the elements that are important to the success of the project. These included the commitment to a shared goal, multi-agency collaboration, and a platform for knowledge exchange. Finally, respondents perceived benefits of the project at both the individual and community level. CONCLUSION: Our project sheds light on many of the practical considerations and challenges associated with a designing and implementing a community-based family intervention project. Community stakeholders input provided important information on their perceived benefits and barriers and can inform and improve future development of community-based family intervention programs.

Development and Evaluation of a Train-the-Trainer Workshop for Hong Kong Community Social Service Agency Staff.

INTRODUCTION: Capacity building approaches are useful in large-scale community-based health promotion interventions. However, models to guide and evaluate capacity building among social service agency staff in community settings are rare in the literature. This paper describes the development and evaluation of a 1-day (7 h) train-the-trainer (TTT) workshop for the "Enhancing Family Well-Being Project". The workshop aimed at equipping staff from different community agencies with the knowledge and skills to design, implement, and evaluate positive psychology-based interventions for their clients in Sham Shui Po, an over-crowded and low-income district in Hong Kong. METHODS: The current TTT extended and improved on our previous successful model by adding research and evaluation methods (including the Logic Model, process evaluation, and randomized controlled trial), which are important to plan and evaluate the community interventions. Evaluation of the TTT was guided by the Integrated Model of Training Evaluation and Effectiveness (IMTEE), with quantitative and qualitative methods. Quantitative data were collected from pretraining (T1), post-training (T2), and 6-month (T3) and 12-month (T4) follow-up surveys. Qualitative data were collected from four focus groups of agency staff after the intervention. RESULTS: Ninety-three staff from 30 community agencies attended the training, and 90 completed the baseline survey. Eighty-eight, 63, and 57 staff performed the evaluations at T2, T3, and T4, respectively. Agency staff were satisfied with the TTT. Immediate enhancement of knowledge, self-efficacy, and positive attitudes toward the training content was found at T2 (Cohen's d ranged from 0.24 to 1.22, all p < 0.05). Enhancement of knowledge of all training contents persisted at T3 and T4 (Cohen's d ranged from 0.34 to 0.63, all p < 0.05). Enhancement of self-efficacy in the use of positive psychology in intervention design persisted at T3 (Cohen's d = 0.22, p = 0.04). The skills learned were utilized to plan and develop subsequent interventions. Twenty-nine interventions were successfully designed and implemented by the agency staff, and delivered to 1,586 participants. The agency staff indicated their intention to utilize the skills they had learned for other interventions (score ≥4 out of 6) and to share these skills with their colleagues. Qualitative feedbacks from 23 agency staff supported the quantitative results. CONCLUSION: Our brief TTT was effectively delivered to a large number of agency staff and showed effects that persisted up to 12 months. Our training and evaluation models may offer a template for capacity building among social service agency staff for community brief, universal family health promotion interventions in diverse settings.

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase.

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3' aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA(Leu) (anticodon 5'-CAG-3') isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA(Leu) (CAG)-a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)-that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.

Structural insights into recognition of MDC1 by TopBP1 in DNA replication checkpoint control.

Activation of the DNA replication checkpoint by the ATR kinase requires protein interactions mediated by the ATR-activating protein, TopBP1. Accumulation of TopBP1 at stalled replication forks requires the interaction of TopBP1 BRCT5 with the phosphorylated SDT repeats of the adaptor protein MDC1. Here, we present the X-ray crystal structures of the tandem BRCT4/5 domains of TopBP1 free and in complex with a MDC1 consensus pSDpT phosphopeptide. TopBP1 BRCT4/5 adopts a variant BRCT-BRCT packing interface and recognizes its target peptide in a manner distinct from that observed in previous tandem BRCT- peptide structures. The phosphate-binding pocket and positively charged residues in a variant loop in BRCT5 present an extended binding surface for the negatively charged MDC1 phosphopeptide. Mutations in this surface reduce binding affinity and recruitment of TopBP1 to γH2AX foci in cells. These studies reveal a different mode of phosphopeptide binding by BRCT domains in the DNA damage response.

53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark.

53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.

Tandem protein interaction modules organize the ubiquitin-dependent response to DNA double-strand breaks.

The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response.

Molecular insights into the function of RING finger (RNF)-containing proteins hRNF8 and hRNF168 in Ubc13/Mms2-dependent ubiquitylation.

The repair of DNA double strand breaks by homologous recombination relies on the unique topology of the chains formed by Lys-63 ubiquitylation of chromatin to recruit repair factors such as breast cancer 1 (BRCA1) to sites of DNA damage. The human RING finger (RNF) E3 ubiquitin ligases, RNF8 and RNF168, with the E2 ubiquitin-conjugating complex Ubc13/Mms2, perform the majority of Lys-63 ubiquitylation in homologous recombination. Here, we show that RNF8 dimerizes and binds to Ubc13/Mms2, thereby stimulating formation of Lys-63 ubiquitin chains, whereas the related RNF168 RING domain is a monomer and does not catalyze Lys-63 polyubiquitylation. The crystal structure of the RNF8/Ubc13/Mms2 ternary complex reveals the structural basis for the interaction between Ubc13 and the RNF8 RING and that an extended RNF8 coiled-coil is responsible for its dimerization. Mutations that disrupt the RNF8/Ubc13 binding surfaces, or that truncate the RNF8 coiled-coil, reduce RNF8-catalyzed ubiquitylation. These findings support the hypothesis that RNF8 is responsible for the initiation of Lys-63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168.

OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function.

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

BRCT domains: easy as one, two, three.

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero- domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.

ATR autophosphorylation as a molecular switch for checkpoint activation.

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.

Molecular basis of BACH1/FANCJ recognition by TopBP1 in DNA replication checkpoint control.

The diverse roles of TopBP1 in DNA replication and checkpoint signaling are associated with the scaffolding ability of TopBP1 to initiate various protein-protein interactions. The recognition of the BACH1/FANCJ helicase by TopBP1 is critical for the activation of the DNA replication checkpoint at stalled replication forks and is facilitated by the C-terminal tandem BRCT7/8 domains of TopBP1 and a phosphorylated Thr(1133) binding motif in BACH1. Here we provide the structural basis for this interaction through analysis of the x-ray crystal structures of TopBP1 BRCT7/8 both free and in complex with a BACH1 phospho-peptide. In contrast to canonical BRCT-phospho-peptide recognition, TopBP1 BRCT7/8 undergoes a dramatic conformational change upon BACH1 binding such that the two BRCT repeats pivot about the central BRCT-BRCT interface to provide an extensive and deep peptide-binding cleft. Additionally, we provide the first structural mechanism for Thr(P) recognition among BRCT domains. Together with systematic mutagenesis studies, we highlight the role of key contacts in governing the unique specificity of the TopBP1-BACH1 interaction.

BACH1/FANCJ acts with TopBP1 and participates early in DNA replication checkpoint control.

Human TopBP1 plays a critical role in the control of DNA replication checkpoint. In this study, we report a specific interaction between TopBP1 and BACH1/FANCJ, a DNA helicase involved in the repair of DNA crosslinks. The TopBP1/BACH1 interaction is mediated by the very C-terminal tandem BRCT domains of TopBP1 and S phase-specific phosphorylation of BACH1 at Thr 1133 site. Interestingly, we demonstrate that depletion of TopBP1 or BACH1 attenuates the loading of RPA on chromatin. Moreover, both TopBP1 and BACH1 are required for ATR-dependent phosphorylation events in response to replication stress. Taken together, our data suggest that BACH1 has an unexpected early role in replication checkpoint control. A specific interaction between TopBP1 and BACH1 is likely to be required for the extension of single-stranded DNA regions and RPA loading following replication stress, which is a prerequisite for the subsequent activation of replication checkpoint.

Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIbeta binding protein 1.

Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.

RAD18 transmits DNA damage signalling to elicit homologous recombination repair.

To maintain genome stability, cells respond to DNA damage by activating signalling pathways that govern cell-cycle checkpoints and initiate DNA repair. Cell-cycle checkpoint controls should connect with DNA repair processes, however, exactly how such coordination occurs in vivo is largely unknown. Here we describe a new role for the E3 ligase RAD18 as the integral component in translating the damage response signal to orchestrate homologous recombination repair (HRR). We show that RAD18 promotes homologous recombination in a manner strictly dependent on its ability to be recruited to sites of DNA breaks and that this recruitment relies on a well-defined DNA damage signalling pathway mediated by another E3 ligase, RNF8. We further demonstrate that RAD18 functions as an adaptor to facilitate homologous recombination through direct interaction with the recombinase RAD51C. Together, our data uncovers RAD18 as a key factor that orchestrates HRR through surveillance of the DNA damage signal.