Calpain 7, a mammalian ortholog of yeast Cpl1 ⁄ Rim13 and fungal PalB, is an atypical calpain that lacks a penta-EF-hand domain. Previously, we reported that a region containing a tandem repeat of microtubule-interacting and transport (MIT) domains in calpain 7 interacts with a subset of endosomal sorting complex required for transport (ESCRT)-III-related proteins, suggesting involvement of calpain 7 in the ESCRT system. Although yeast and fungal calpains are thought to be involved in alkaline adaptation via limited proteolysis of specific transcription factors, proteolytic activity of calpain 7 has not been demonstrated yet. In this study, we investigated the interaction between calpain 7 and a newly reported ESC-RT-III family member, increased sodium tolerance-1 (IST1), which possesses two different types of MIT-interacting motifs (MIM1 and MIM2). We found that glutathione-S-transferase (GST)-fused tandem MIT domains of calpain 7 (calpain 7MIT) pulled down FLAG-tagged IST1 expressed in HEK293T cells. Coimmunoprecipitation assays with various deletion or point mutants of epitope-tagged calpain 7 and IST1 revealed that both repetitive MIT domains and MIMs are required for efficient interaction. Direct MIT-MIM binding was confirmed by a pulldown experiment with GST-fused IST1 MIM and purified recombinant calpain 7MIT. Furthermore, we found that the GST-MIM protein enhances the autolysis of purified Strep-tagged monomeric green fluorescent protein (mGFP)-fused calpain 7 (mGFP-calpain 7-Strep). The autolysis was almost completely abolished by 10 mm N-ethylmaleimide but only partially inhibited by 1 mm leupeptin or E-64. The putative catalytic Cys290-substituted mutant (mGFP-calpain 7 C290S -Strep) showed no autolytic activity. These results demonstrate for the first time that human calpain 7 is proteolytically active, and imply that calpain 7 is activated in the ESCRT system. by pull down (MI:0096)CBB, Coomassie Brilliant Blue R-250; CHMP, charged multivesicular body protein; CSD1, calpastatin domain 1; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent protein; GST, glutathione-S-transferase; IST1, increased sodium tolerance-1; mGFP, monomeric green fluorescent protein; MIM, microtubule-interacting and transport-interacting motif; MIT, microtubule-interacting and transport; pAb, polyclonal antibody; VPS, vacuolar protein sorting; WB, western blot.

The name calpain was historically given to a protease that is activated by Ca2+ and whose primary structure contains a Ca2+‐binding penta‐EF‐hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2‐domain‐like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain‐like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain‐7, indicating that the calpain‐7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N‐terminal region of calpain‐7 has a tandem repeat of microtubule‐interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca2+‐binding proteins including ALG‐2 that associates with ALIX (an ESCRT‐III accessory protein) and TSG101 (an ESCRT‐I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein–protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system.

Sickle cell disease (SCD) is a heritable disorder caused by β-globin gene mutations. Induction of fetal γ-globin is an established therapeutic strategy. Recently, epigenetic modulators, including G9a inhibitors, have been proposed as therapeutic agents. However, the molecular mechanisms whereby these small molecules reactivate γ-globin remain unclear. Here we report the development of a highly selective and non-genotoxic G9a inhibitor, RK-701. RK-701 treatment induces fetal globin expression both in human erythroid cells and in mice. Using RK-701, we find that BGLT3 long non-coding RNA plays an essential role in γ-globin induction. RK-701 selectively upregulates BGLT3 by inhibiting the recruitment of two major γ-globin repressors in complex with G9a onto the BGLT3 gene locus through CHD4, a component of the NuRD complex. Remarkably, BGLT3 is indispensable for γ-globin induction by not only RK-701 but also hydroxyurea and other inducers. The universal role of BGLT3 in γ-globin induction suggests its importance in SCD treatment.

DDHD2/KIAA0725p is a mammalian intracellular phospholipase A1 that exhibits phospholipase and lipase activities. Mutation of the DDHD2 gene causes hereditary spastic paraplegia (SPG54), an inherited neurological disorder characterized by lower limb spasticity and weakness. Although previous studies demonstrated lipid droplet accumulation in the brains of SPG54 patients and DDHD2 knockout mice, the cause of SPG54 remains elusive. Here, we show that ablation of DDHD2 in mice induces age-dependent apoptosis of motor neurons in the spinal cord. In vitro, motor neurons and embryonic fibroblasts from DDHD2 knockout mice fail to survive and are susceptible to apoptotic stimuli. Chemical and probe-based analysis revealed a substantial decrease in cardiolipin content and an increase in reactive oxygen species generation in DDHD2 knockout cells. Reactive oxygen species production in DDHD2 knockout cells was reversed by the expression of wild-type DDHD2, but not by an active-site DDHD2 mutant, DDHD2 mutants related to hereditary spastic paraplegia, or DDHD1, another member of the intracellular phospholipase A1 family whose mutation also causes spastic paraplegia (SPG28). Our results demonstrate the protective role of DDHD2 for mitochondrial integrity and provide a clue to the pathogenic mechanism of SPG54.

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.