Cullin-RING finger ligases (CRLs) 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, CUL4 associates with 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 (DCAF) 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 AP-MS 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-SILAC 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.

Defining the molecular phenotype of single cells in-situ is key for understanding tissue architecture in health and disease. Advanced imaging platforms have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. Furthermore, high-precision laser microdissection of tissue on membrane glass slides is a powerful method for spatial omics technologies and single-cell type spatial proteomics in particular. However, current histology protocols have not been compatible with glass membrane slides and laser microdissection for automated staining platforms and routine histology procedures. This has prevented the combination of advanced staining procedures with laser microdissection. In this study we describe a novel method for handling glass membrane slides that enables automated, eight-color multiplexed immunofluorescence staining and high-quality imaging followed by precise laser-guided extraction of single cells. The key advance is the glycerol-based modification of heat-induced epitope retrieval protocols, termed 'G-HIER'. We find that this altered antigen-retrieval solution prevents membrane distortion. Importantly, G-HIER is fully compatible with current antigen retrieval workflows and mass spectrometry-based proteomics and does not affect proteome depth or quality. To demonstrate the versatility of G-HIER for spatial proteomics, we apply the recently introduced Deep Visual Proteomics technology to perform single-cell type analysis of adjacent suprabasal and basal keratinocytes of human skin. G-HIER overcomes previous incompatibility of standard and advanced staining protocols with membrane glass slides and enables robust integration with routine histology procedures, high-throughput multiplexed imaging and sophisticated downstream spatial omics technologies.

Uterine environment is tightly and finely regulated via various signalling pathways mediated through endocrine, exocrine, autocrine, juxtracrine and paracrine mechanisms. In utero signalling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo and even inter-kingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signalling and communications. Interestingly, the changes in UF-EV proteome during the bovine oestrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass-spectrometry was employed to compare UF-EV proteomes at day 0, 7 and 16 of the oestrous cycle to understand the oestrous cycle dependant dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. 159 UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (P ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the oestrous cycle. Interestingly, comparison of hormone synchronized EV-proteomes to natural cycle UF-EVs indicated shift of signalling. Finally, UF-EVs can be used to improve embryo production in vitro.

Cell metabolism generates numerous intermediate metabolites that could serve as feedback and feed-forward regulation substances for post-translational modification. Lactate, a metabolic product of glycolysis, has recently been conceptualized to play a pleiotropic role in shaping cell identities through metabolic rewiring and epigenetic modifications. Lactate-derived carbons, sourced from glucose, mediate the crosstalk among glycolysis, lactate and lactylation. Furthermore, the multiple metabolic fates of lactate make it an ideal substrate for metabolic imaging in clinical application. Several studies have identified the crucial role of protein lactylation in human diseases associated with cell fate determination, embryonic development, inflammation, neoplasm and neuropsychiatric disorders. Herein, this review will focus on the metabolic fate of lactate-derived carbon to provide useful information for further research and therapeutic approaches in human diseases. We comprehensively discuss its role in reprogramming and modification during the regulation of glycolysis, the clinical translation prospects of the hyperpolarized lactate signal, lactyl modification in human diseases, and its application with other techniques and omics.

We present an integrated immunopeptidomics and proteomics study of SARS-CoV-2 infection to comprehensively decipher the changes in host cells in response to viral infection. Immunopeptidomics analysis identified viral antigens presented by host cells through both Class I and Class II MHC system for recognition by the adaptive immune system. The host proteome changes were characterized by quantitative proteomics and glycoproteomics and from these data, the activation of TLR3-interferon pathway was identified. Glycosylation analysis of HLA proteins from the elution and flow-through of immunoprecipitation revealed that SARS-CoV-2 infection changed the glycosylation pattern of certain HLA alleles with different HLA alleles showing distinct dynamic changes in relative abundance. The difference in the glycosylation and abundance of HLA alleles changed the number of strong binding antigens each allele presented, suggesting the impact of SARS-CoV-2 infection on antigen presentation is allele specific. These results could be further exploited to explain the imbalanced response from innate and adaptive immune system in severe COVID-19 cases, which would be helpful for the development of therapeutics and vaccine for COVID-19 and preparation for future pandemic.