Pulse-SILAC and Interactomics Reveal Distinct DDB1-CUL4-Associated Factors, Cellular Functions, and Protein Substrates.

Cullin-RING finger ligases represent the largest family of ubiquitin ligases. They are responsible for the ubiquitination of ∼20% of cellular proteins degraded through the proteasome, by catalyzing the transfer of E2-loaded ubiquitin to a substrate. Seven cullins are described in vertebrates. Among them, cullin 4 (CUL4) associates with DNA damage-binding protein 1 (DDB1) to form the CUL4-DDB1 ubiquitin ligase complex, which is involved in protein ubiquitination and in the regulation of many cellular processes. Substrate recognition adaptors named DDB1/CUL4-associated factors (DCAFs) mediate the specificity of CUL4-DDB1 and have a short structural motif of approximately forty amino acids terminating in tryptophan (W)-aspartic acid (D) dipeptide, called the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested that at least sixty WD40-containing proteins could act as adaptors for the DDB1/CUL4 complex. To better define this association and classification, the interaction of each DCAFs with DDB1 was determined, and new partners and potential substrates were identified. Using BioID and affinity purification-mass spectrometry approaches, we demonstrated that seven WD40 proteins can be considered DCAFs with a high confidence level. Identifying protein interactions does not always lead to identifying protein substrates for E3-ubiquitin ligases, so we measured changes in protein stability or degradation by pulse-stable isotope labeling with amino acids in cell culture to identify changes in protein degradation, following the expression of each DCAF. In conclusion, these results provide new insights into the roles of DCAFs in regulating the activity of the DDB1-CUL4 complex, in protein targeting, and characterized the cellular processes involved.

A Comprehensive Analysis of Skin Cancer Concerns and Protective Practices in Manitoba, Canada, Highlights Lack of Skin Cancer Awareness and Predominance of High-Risk Sun Exposure Behaviors.

The rapidly increasing skin cancer rates in Canada are alarming, with current data estimating that 1/3 of Canadians will be affected in their lifetime. Thus, deeper understanding of high-risk sun exposure behaviors is needed to help counter this trend. Only limited action has been taken by federal/provincial governments to reduce skin cancer incidence. A cross-sectional survey study was conducted in Manitoba, with frequency counts, means, and percentages used to encapsulate responses. Age- and gender-adjusted odds ratios were calculated using logistic regression analyses. Our study identified worrying inadequacies in sun protective behaviors and attitudes, with the threat of such high-risk behaviors amplified by a lack of skin cancer awareness. Alarming elements were noted in participants' sun exposure history (>65% reported a history of sunburns, >50% previously used a tanning bed, and >75% recently tanned for pleasure), beliefs and attitudes (>50% believe that they look better/healthier with a tan, and >40% believe that having a base tan is protective against further sun damage), and sun protection efforts (sun protective clothing was used <60% of the time, sunscreen was used by <50%, and there was a lack of knowledge about sunscreen characteristics in ~30% of respondents), in addition to significant differences being established between demographic subgroups (based on gender, age, skin phototype, income, and education attained). This study provides worrisome insight onto the grim landscape of sun protective behaviors and attitudes in Manitoba, which will inevitably translate into higher skin cancer rates and should serve as a call to action to promote targeted public health messaging in this jurisdiction and beyond.

Destabilization of the MiniChromosome Maintenance (MCM) complex modulates the cellular response to DNA double strand breaks.

DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is replicated only once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both the initiation and elongation of DNA replication. Although only two DNA helicase activities are necessary to establish a bidirectional replication fork from each replication origin, a large excess of MCM complexes is amassed and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most are displaced from the DNA during the S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of a reduction in MCM complex. The alteration of MCM proteins induced a change in the activation of key factors of the DDR in response to Etoposide treatment. Etoposide-induced DNA damage affected the phosphorylation of γ-H2AX, CHK1 and CHK2 without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we identified a decrease in both HR and NHEJ associated with a decrease in MCM complex.