AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex in which the ubiquitin-like (UBL) domains of SHARPIN and HOIL-1L interact with HOIP to determine the structural stability of LUBAC. The interactions between subunits within LUBAC have been a topic of extensive research. However, the impact of the LTM motif on the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP remains unclear. Here, we discover that the absence of the LTM motif in the AlphaFold2-predicted LUBAC structure alters the HOIP-UBA structure. We employ GeoPPI to calculate the changes in binding free energy (ΔG) caused by single-point mutations between subunits, simulating their protein-protein interactions. The results reveal that the presence of the LTM motif decreases the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP, leading to a decrease in the structural stability of LUBAC. Furthermore, using the AlphaFold2-predicted results, we find that HOIP (629‒695) and HOIP-UBA bind to both sides of HOIL-1L-UBL, respectively. The experiments of Gromacs molecular dynamics simulations, SPR and ITC demonstrate that the elongated domain formed by HOIP (629‒695) and HOIP-UBA, hereafter referred to as the HOIP (466‒695) structure, interacts with HOIL-1L-UBL to form a structurally stable complex. These findings illustrate the collaborative interaction between HOIP-UBA and HOIP (629‒695) with HOIL-1L-UBL, which influences the structural stability of LUBAC.

Biochemical and functional characterization of the N-terminal ubiquitin-like domain of human SHARPIN.

SHARPIN, an accessory subunit of the E3 ligase complex LUBAC, participates in the formation of LUBAC through the ubiquitin-like (UBL) domain located in the central region of SHARPIN and interacts with the ubiquitin-associated domain (UBA) of the catalytic subunit HOIP. However, the role of the N-terminal UBL domain of SHARPIN in stable LUBAC formation has not been clarified. In this study, the 1-127 domain, 128-309 domain, and UBL domain of SHARPIN expression vectors were constructed using the molecular biology method. Then the co-expression of SUMO fusion protein combined with SUMO protease (ULP enzyme) in Escherichia coli was successfully applied to improve the soluble expression of target protein. The results of circular dichroism proved that they all belong to the α+ß class of proteins. The results of size exclusion chromatography showed that 128-309 domain could combine with HOIP and HOIL-1L to participate in the stability of LUBAC. Both thermal-induced and urea-induced unfolding experiment results demonstrated that the existence of the N-terminal UBL domain could make the overall structure more stable than the alone UBL domain. Biosensor experiments indicated that the existence of the N-terminal UBL domain strengthened the binding ability of the UBL domain and the UBA domain. These results were conducive to further study the structure and function of SHARPIN.

Effects of removing a highly conserved disulfide bond in ubiquitin-associated domain of human HOIP on biochemical characteristics.

Disulfide bond formed between the cysteine pairs plays a key role in maintaining the integrity of the protein structure and function. The ubiquitin-associated (UBA) domain of human HOIP contains three cysteine residues, Cys504, Cys551, and Cys572. Disulfide bonds formed by Cys504 and Cys551 residues are highly conserved, but the effect of disulfide bonds on the biochemical characteristics of UBA has not been elucidated. In addition, due to the presence of isolated Cys572, inactive inclusion bodies may be formed during protein expression or trigger protein aggregation during protein purification. In this study, the co-expression of SUMO fusion protein combined with SUMO protease (ULP enzyme) in Escherichia coli was successfully applied to improve the soluble expression of UBA domain. Introduced three mutants (UBAC551A, UBAC572A and UBAC551,572A) determined the effects of disulfide bonds on the biochemical characteristics of UBA. Circular dichroism and analytical size exclusion chromatography results showed that the target proteins obtained by co-expression could be folded correctly and had biological activity. Both thermal-induced and urea-induced results demonstrated that the elimination of disulfide bonds would significantly reduce the stability of UBA. Fluorescence spectroscopy result showed that the elimination of disulfide bonds slightly increases the binding affinity of UBA to ligands. In summary, soluble, stable and active UBA domain and its mutants were prepared by co-expression system, which will further contribute to the structural and functional research of UBA.

Continuous biomass and lipid production from local chlorella-bacteria consortium in raw wastewater using volatile fatty acids.

OBJECTIVES: To evaluate mixotrophic cultivation of microalgae-bacteria consortium in raw wastewater by stepwise addition of fermented effluent containing volatile fatty acids (VFAs). RESULTS: Stepwise increase of VFAs enhanced algal biomass and lipid production, ammonia and phosphate removals. The highest biomass and lipid yield were 1.94 g L-1 and 310 mg L-1 when the addition of fermented effluent containing VFAs increased to 30% (v/v). With the same cultivation conditions, the maximum removals efficiency of ammonia and phosphate were 26.4 and 11.3 mg L-1 d-1. Bacterial diversities increased with the increasing concentration of VFAs and their communities were identified as phyla Actinobacteria, Bacteroidetes, Cyanobacteria and Proteobacteria. CONCLUSIONS: Although bacterial quantities increased with algae growth concurrently, the objective of culturing microalgae-bacteria consortium in raw wastewater without sterilization to produce biomass and lipid yield still can be realized.