Plant metacaspases type I (MCA-Is), the closest structural homologs of caspases, are key proteases in stress-induced regulated cell death processes in plants. However, no plant MCA-Is have been characterized in vitro to date. Here, we show that only plant MCA-Is contain a highly hydrophobic loop within the C terminus of their p10 domain. When removed, soluble and proteolytically active plant MCA-Is can be designed and recombinantly produced. We show that the activity of MCA-I depends on calcium ions and that removal of the hydrophobic loop does not affect cleavage and covalent binding to its inhibitor SERPIN. This novel approach will finally allow the development of tools to detect and manipulate the activity of these cysteine proteases in vivo and in planta .

Cellular double-stranded RNA-binding proteins (DRBPs) play important roles in the regulation of innate immune responses and microRNA (miRNA) biogenesis. The current study aimed to understand whether OV20.0, a DRBP of orf virus (ORFV), is involved in cellular RNA biogenesis via association with host DRBPs. We found that OV20.0 interacts with DiGeorge syndrome critical region 8 (DGCR8), a subunit of the miRNA processor complex, and binds to primary- and precursor-miRNA. Additionally, OV20.0 regulates DGCR8 expression in multiple ways, including through interaction with the DGCR8 protein and binding to DGCR8 mRNA. Lastly, our data show that DGCR8 plays an antiviral role against ORFV infection, whereas it is beneficial for influenza virus propagation, indicating that the underlying mechanisms could be diverse among different viruses.

Transient receptor potential vanilloid subfamily 1 (TRPV1) is a non-selective cation channel protein expressed in neuronal and non-neuronal cells. Although TRPV1 is implicated in thermogenesis and diet-induced obesity (DIO), its precise role remains controversial. TRPV1 −/− mice are protected from DIO, while TRPV1 activation enhances thermogenesis to prevent obesity. To reconcile this, we fed wild-type and TRPV1 −/− mice for 32 weeks with normal chow or a high-fat diet and analyzed the weight gain, metabolic activities, and thermogenic protein expression in white and brown fats. TRPV1 −/− mice became obese, exhibited reduced locomotor activity, reduced energy expenditure, enhanced hepatic steatosis, and decreased thermogenic protein expression in adipose tissues. Our data reveal that lack of TRPV1 does not prevent obesity, but rather enhances metabolic dysfunction.

To disentangle the elusive lipid–protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

Brown and beige adipocytes dissipate energy by uncoupling protein 1 (UCP1)-dependent and UCP1-independent thermogenesis, which may be utilized to develop treatments against obesity. We have found that mRNA and protein expression of the alanine/serine/cysteine transporter-1 (ASC-1) was induced during adipocyte differentiation of human brown-prone deep neck and beige-competent subcutaneous neck progenitors, and SGBS preadipocytes. cAMP stimulation of differentiated adipocytes led to elevated uptake of serine, cysteine, and glycine, in parallel with increased oxygen consumption, augmented UCP1-dependent proton leak, increased creatine-driven substrate cycle-coupled respiration, and upregulation of thermogenesis marker genes and several respiratory complex subunits; these outcomes were impeded in the presence of the specific ASC-1 inhibitor, BMS-466442. Our data suggest that ASC-1-dependent consumption of serine, cysteine, and glycine is required for efficient thermogenic stimulation of human adipocytes.

Microtubule nucleation is mainly mediated by the γ-tubulin ring complex (γTuRC), whose core components are γ-tubulin and γ-tubulin complex proteins GCP2–6. A substantial fraction of γ-tubulin also exists with GCP2 and GCP3 in a tetramer called the γ-tubulin small complex (γTuSC). To date, the mechanisms underlying the turnover of γ-tubulin and GCPs have remained unclear. Here, we show that γ-tubulin, GCP2, and GCP3 are proteolyzed by the ubiquitin-proteasome system, and we identify cullin 1, cullin 4A, and cullin 4B as the E3 ligases that mediate the ubiquitination and, consequently, the degradation of γ-tubulin. Notably, we found that γTuSC disassembly promotes the degradation of γ-tubulin, GCP2, and GCP3, which indicates a role for γTuSCs in the stabilization of its components.