Commentary on: Calpain-2 participates in the process of calpain-1 inactivation

Calpain belongs to the calcium-dependent non-lysosomal cysteine protease. Calpain-1 (C1) and calpain-2 (C2) expression are ubiquitous in mammals and an important mediator of the action of calcium. Specific substrate cleavage by C1 and C2 is critical for several calcium-dependent cellular pathways including neuronal function, muscle contraction, signal transduction, cell differentiation, proliferation, and apoptosis. Research suggests that C1 and C2 perform similar functions due to their structurally highly similar isoforms. Increasing evidence suggests that C1 and C2 carry out their specific function in vivo. A recent paper published by Shinkai-Ouchi et al. (Bioscience Reports (2020) 40, DOI: 10.1042/BSR20200552) elucidated the mechanism to differentiate the function of each calpain with respect to the efficiency and longevity for proteolysis after activation. Further, the study represented that C1 and C2 do not synergistically perform their work in vitro. On the other hand, the activity of C1 is reduced in presence of C2. This insight establishes the platform for future studies to examine how C2 regulates the C1 for substrate proteolysis.     

Single-molecule photobleaching: Instrumentation and applications

Single-molecule photobleaching (smPB) technique is a powerful tool for characterizing molecular assemblies. It can provide a direct measure of the number of monomers constituting a given oligomeric particle and generate the oligomer size distribution in a specimen. A major current application of this technique is in understanding protein aggregation, which is linked to many incurable diseases. Quantitative measurement of the size distribution of an aggregating protein in a physiological solution remains a difficult task, since techniques such as dynamic light scattering or fluorescence correlation spectroscopy (FCS) can provide an average size, but cannot accurately resolve the underlying size distribution. Here we describe the smPB method as implemented on a home-built total internal reflection fluorescence microscope (TIRF). We first describe the construction of a TIRF microscope, and then demonstrate the power of smPB by characterizing a solution of Amylin (hIAPP) oligomers, a 37-residue peptide whose aggregation is associated with Type II diabetes. We compare our results with FCS data obtained from the same specimen, and discuss the advantages and disadvantages of the two techniques.     

miR-125b promotes cell death by targeting spindle assembly checkpoint gene MAD1 and modulating mitotic progression

The spindle assembly checkpoint (SAC) is a ‘wait-anaphase'' mechanism that has evolved in eukaryotic cells in response to the stochastic nature of chromosome–spindle attachments. In the recent past, different aspects of the SAC regulation have been described. However, the role of microRNAs in the SAC is vaguely understood. We report here that Mad1, a core SAC protein, is repressed by human miR-125b. Mad1 serves as an adaptor protein for Mad2 – which functions to inhibit anaphase entry till the chromosomal defects in metaphase are corrected. We show that exogenous expression of miR-125b, through downregulation of Mad1, delays cells at metaphase. As a result of this delay, cells proceed towards apoptotic death, which follows from elevated chromosomal abnormalities upon ectopic expression of miR-125b. Moreover, expressions of Mad1 and miR-125b are inversely correlated in a variety of cancer cell lines, as well as in primary head and neck tumour tissues. We conclude that increased expression of miR-125b inhibits cell proliferation by suppressing Mad1 and activating the SAC transiently. We hypothesize an optimum Mad1 level and thus, a properly scheduled SAC is maintained partly by miR-125b.     

L-Phenylalanine production by double auxotrophic analogue resistant mutants of Arthrobacter globiformis

A number of tryptophan plus tyrosine double auxotrophic mutants isolated by the NTG treatment of a glutamate producing strain of Arthrobacter globiformis were found to excrete phenylalanine in a mineral salt medium. By controlling the pH of the medium to near neutrality, the active growth period could be extended up to 72 h and more phenylalanine was accumulated compared to the unregulated culture where the growth period took up to 48 h. Under optimum culture conditions, the best double auxotroph (TT-39) produced 3 g phenylalanine/l. Further improvement of phenylalanine production has been achieved by the step-by-step isolation of a mutant resistant to the phenylalanine analogues p-fluorophenylalanine (PFP) and β-2-thienylalanine (TA) from the TT-39 strain. Under optimum culture conditions, the best double auxotrophic analogue resistant mutant TT-39 PT^r-21 yielded 8.7 g/l phenylalanine.