Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane–localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD–CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce “donut” mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1–CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1–CL interactions in stress-induced mitochondrial fission.
Aims: Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach.
Methods and Results: The significant factors influencing the protease production as screened by Plackett–Burman method were identified as soybean flour and FeCl3. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l−1): NaCl, 250; KCl, 2; MgSO4, 10; tri‐Na‐citrate, 1·5; soybean flour, 10 and FeCl3, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml−1 of protease.
Conclusions: Soybean flour and FeCl3 were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9‐fold increase in extracellular protease production.
Significance and Impact of the Study: The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.
Cardiac myosin binding
protein C (cMyBPC) is a critical multidomain
protein that modulates myosin cross bridge behavior and cardiac contractility.
cMyBPC is principally regulated by phosphorylation of the residues
within the M-domain of its N-terminus. However, not much is known
about the phosphorylation or other post-translational modification
(PTM) landscape of the central C4C5 domains. In this study, the presence
of phosphorylation outside the M-domain was confirmed in vivo using
mouse models expressing cMyBPC with nonphosphorylatable serine (S)
to alanine substitutions. Purified recombinant mouse C4C5 domain constructs
were incubated with 13 different kinases, and samples from the 6 strongest
kinases were chosen for mass spectrometry analysis. A total of 26
unique phosphorylated peptides were found, representing 13 different
phosphorylation sites including 10 novel sites. Parallel reaction
monitoring and subsequent mutagenesis experiments revealed that the
S690 site (UniProtKB O70468) was the predominant target of PKA and
PKG1. We also report 6 acetylation and 7 ubiquitination sites not
previously described in the literature. These PTMs demonstrate the
possibility of additional layers of regulation and potential importance
of the central domains of cMyBPC in cardiac health and disease. Data
are available via ProteomeXchange with identifier PXD031262.
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