Lrrk2, a gene linked to Parkinson’s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson’s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

The history of protein kinase CK2 is crowded with paradoxes and unanticipated findings. Named after a protein (casein) that is not among its physiological substrates, CK2 remained in search of its targets for more than two decades after its discovery in 1954, but it later came to be one of the most pleiotropic protein kinases. Being active in the absence of phosphorylation and/or specific stimuli, it looks unsuitable to participate in signaling cascades, but its “lateral” implication in a variety of signaling pathways is now soundly documented. At variance with many “onco-kinases”, CK2 is constitutively active, and no oncogenic CK2 mutant is known; still high CK2 activity correlates to neoplasia. Its pleiotropy and essential role may cast doubts on the actual “druggability” of CK2; however, a CK2 inhibitor is now in Phase II clinical trials for the treatment of cancer, and cell clones viable in the absence of CK2 are providing information about the mechanism by which cancer becomes addicted to high CK2 levels. A phosphoproteomics analysis of these CK2 null cells suggests that CK2 pleiotropy may be less pronounced than expected and supports the idea that the phosphoproteome generated by this kinase is flexible and not rigidly pre-determined.

Heart failure is the worst epilogue of all cardiovascular diseases and still represents nowadays the leading cause of mortality with no effective clinical treatments, apart from organ transplantation with allogeneic or artificial substitutes. Although applied as gold standard, allogeneic heart transplantation cannot be considered a permanent clinical answer due to several drawbacks, as the side effects of administered immunosuppressive therapies. For the increasing number of heart failure patients, a biological cardiac substitute based on a decellularized organ and autologous cells might be the lifelong, biocompatible solution free from the need for immunosuppression regimen. A novel decellularization method is here proposed and tested on rat hearts in order to reduce the concentration and incubation time with cytotoxic detergents needed to render acellular these organs. By protease inhibition, anti-oxidation and excitation-contraction uncoupling in simultaneous perfusion/submersion modality, a strongly limited exposure to detergents was sufficient to generate very well-preserved acellular hearts with unaltered extracellular matrix macro- and microarchitecture, as well as bioactivity.

Cheese production is an applied biotechnology whose proper outcome relies strictly on the complex interactive dynamics which unfold within defined microbial groups. These may start being active from the collection of milk and continue up to its final stages of maturation. One of the critical parameters playing a major role is the milk refrigeration temperature before pasteurization as it can affect the proportion of psychrotrophic taxa abundance in the total milk bacterial population. While a standard temperature of 4 °C is the common choice, due to its general growth control effect, it does have a potential drawback. This is due to the fact that some cold-tolerant genera present a proteolytic activity with uncompleted proliferation, which could negatively affect curd clotting and regular cheese maturation. Moreover, accidental thermal variations of milk before cheese-making, in a plus or minus direction, can occur both at farm collection sites and during transfer to dairy plant. This present research, directly commissioned by a major fresh cheese-producing company, includes an in-factory trial. In this trial, a gradient of temperatures from 4 °C to 13 °C, which were subsequently reversed, was purposely adopted to: (a) verify sensory alterations in the resulting product at different maturation stages, and, (b) analyze, in parallel, using DNA extraction and 16S-metabarcoding sequencing from the same samples, the presence, abundance and corresponding taxonomical identity of all the bacteria featured in communities found in milk and cheese samples. Overall, 1,714 different variants were detected and sorted into 394 identified taxa. Significant bacterial community shifts in cheese were observed in response to milk refrigeration temperature and subsequently associated with samples having altered scores in sensory panel tests. In particular, proteolytic psychrotrophes were outcompeted by Enterobacteriales and by other taxa at the peak temperature of 13 °C, but aggressively increased in the descent phases, upon the cooling down of milk to values of 7 °C. Relevant clues have been collected for better anticipation of thermal abuse effects or parameter variations allowing for improved handling of technical processing conditions by the cheese manufacturing industry.

Despite recent advances in the development of BRAF kinase inhibitors (BRAFi) for BRAF-mutant melanomas, development of resistance remains a major clinical problem. In addition to genetic alterations associated with intrinsic resistance, several adaptive response mechanisms are known to be rapidly activated to allow cell survival in response to treatment, limiting efficacy. A better understanding of the mechanisms driving resistance is urgently needed to improve the success of BRAF-targeted therapies and to make therapeutic intervention more durable. In this study, we identify the mitogen-activated protein kinase (MAPK) p38 as a novel mediator of the adaptive response of melanoma cells to BRAF-targeted therapy. Our findings demonstrate that BRAFi leads to an early increase in p38 activation, which promotes phosphorylation of the transcription factor SOX2 at Ser251, enhancing SOX2 stability, nuclear localization, and transcriptional activity. Furthermore, functional studies show that SOX2 depletion increases sensitivity of melanoma cells to BRAFi, whereas overexpression of a phosphomimetic SOX2-S251E mutant is sufficient to drive resistance and desensitize melanoma cells to BRAFi in vitro and in a zebrafish xenograft model. We also found that SOX2 phosphorylation at Ser251 confers resistance to BRAFi by binding to the promoter and increasing transcriptional activation of the ATP-binding cassette drug efflux transporter ABCG2. In summary, we unveil a p38/SOX2-mediated mechanism of adaptive response to BRAFi, which provides prosurvival signals to melanoma cells against the cytotoxic effects of BRAFi prior to acquiring resistance.