Mitochondrial Carrier Homolog 2 Functionally Co-operates With BH3 Interacting-Domain Death Agonist in Promoting Ca2+-Induced Neuronal Injury

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic member of the Bcl-2 protein family. While proteolytic processing of BID links death receptor-induced apoptosis to the mitochondrial apoptosis pathway, we previously showed that full length BID also translocates to mitochondria during Ca2+-induced neuronal cell death. Moreover, mitochondrial carrier homolog 2 (MTCH2) was identified as a mitochondrial protein that interacts with BID during cell death. We started our studies by investigating the effect of Mtch2 silencing in a well-established model of Ca2+-induced mitochondrial permeability transition pore opening in non-neuronal HCT116 cells. We found that silencing of Mtch2 inhibited mitochondrial swelling and the associated decrease in mitochondrial energetics, suggesting a pro-death function for MTCH2 during Ca2+-induced injury. Next, we explored the role of BID and MTCH2 in mediating Ca2+-induced injury in primary cortical neurons triggered by prolonged activation of NMDA glutamate receptors. Analysis of intracellular Ca2+ transients, using time-lapse confocal microscopy, revealed that neurons lacking Bid showed markedly reduced Ca2+ levels during the NMDA excitation period. These Ca2+ transients were further decreased when Mtch2 was also silenced. Collectively, our data suggest that BID and MTCH2 functionally interact to promote Ca2+-induced neuronal injury.

PAM-interacting domain swapping is extensively utilized in nature to evolve CRISPR-Cas9 nucleases with altered PAM specificities

It has been anticipated that protospacer adjacent motif (PAM) specificity of the CRISPR-Cas9 nucleases protospacer appears to be modular. Here we present the finding that naturally occurring domain swapping has been extensively involved in altering its PAM specificities. Sequence analysis of streptococcal Cas9 sequences revealed conservation of three distinct PAM-interacting motifs, with phylogenetic analysis of full-length Cas9 and the PID demonstrating that PAM interacting domain (PID) domain swapping was extensively utilized to diversify its PAM specificity. An extended analysis of 582 representative Cas9 sequences revealed that this PIDswapping was broadly present in most of the investigated genera. Mimicking the natural PID domain swapping, a functional chimeric enzyme, based on the scaffold of compact Staphylococcus aureus Cas9, with novel NNAAAA PAM specificity was developed. In summary, our findings shed new light on a rich source of exchangeable PID domains in Cas9 genes, which can be mined for domain swapping aiming to an effective PAM refinement.

Dynamin is primed at endocytic sites for ultrafast endocytosis

SummaryDynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane when the proline-rich domain of this variant is dephosphorylated. When this domain is mutated to include phosphomimetic residues or Syndapin 1’s dynamin-interacting domain is mutated, Dynamin 1xA becomes diffuse, and consequently, ultrafast endocytosis slows down by ∼100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

Hepatoprotective Activity of InlB321/15, the HGFR Ligand of Bacterial Origin, in CCI4-Induced Acute Liver Injury Mice

HGF (hepatocyte growth factor)/HGFR (HGF receptor) signaling pathway is a key pathway in liver protection and regeneration after acute toxic damage. Listeria monocytogenes toxin InlB contains a HGFR-interacting domain and is a functional analog of HGF. The aim of this work was to evaluate the hepatoprotective activity of the InlB HGFR-interacting domain. The recombinant HGFR-interacting domain InlB321/15 was purified from E. coli. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was used to measure InlB321/15 mitogenic activity in HepG2 cells. Activation of MAPK- and PI3K/Akt-pathways was tracked with fluorescent microscopy, Western blotting, and ELISA. To evaluate hepatoprotective activity, InlB321/15 and recombinant human HGF (rhHGF) were intravenously injected at the same concentration of 2 ng·g−1 to BALB/c mice 2 h before liver injury with CCl4. InlB321/15 caused dose-dependent activation of MAPK- and PI3K/Akt-pathways and correspondent mitogenic effects. Both InlB321/15 and rhHGF improved macroscopic liver parameters (liver mass was 1.51, 1.27 and 1.15 g for the vehicle, InlB321/15 and rhHGF, respectively, p < 0.05), reduced necrosis (24.0%, 16.18% and 21.66% of the total area for the vehicle, InlB321/15 and rhHGF, respectively, p < 0.05). Obtained data suggest that InlB321/15 is a promising candidate for a tissue repair agent.