Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

Molecular cloning and biochemical characterization of the phospholipid scramblase SCRM-1 from Caenorhabditis elegans.

In this study, the SCRM-1 gene from Caenorhabditis elegans was cloned and overexpressed in E. coli to study the biochemical properties of scramblase. This is the first report showing that this scramblase from C. elegans possesses a Ca2+-dependent and head group-independent scramblase activity. The SCRM-1 of C.elegans possesses functional domains including a single EF-hand-like Ca2+ binding domain, as human scramblases do. A point mutation in the EF-hand-like Ca2+ binding motif results in loss of scramblase activity. Other biochemical assays like carbocyanine staining, Tb3+ luminescence, Tryptophan fluorescence, and CD spectroscopy strongly proved the role of the EF-hand motif for functional activity. The increase in protein size in solution upon incubating with Ca2+ shows ligand-dependent oligomerization and conformational changes.

Activin E is a TGFß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming Growth Factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, similar to ActC. Collectively, our results establish ActE as an ALK7 ligand, thereby providing a link between genetic and in vivo studies of ActE as a regulator of adipose tissue.

Regulation and functions of membrane lipids: Insights from Caenorhabditis elegans.

The Caenorhabditis elegans plasma membrane is composed of glycerophospholipids and sphingolipids with a small cholesterol. The C. elegans obtain the majority of the membrane lipids by modifying fatty acids present in the bacterial diet. The metabolic pathways of membrane lipid biosynthesis are well conserved across the animal kingdom. In C. elegans CDP-DAG and Kennedy pathway produce glycerophospholipids. Meanwhile, the sphingolipids are synthesized through a different pathway. They have evolved remarkably diverse mechanisms to maintain membrane lipid homeostasis. For instance, the lipid bilayer stress operates to accomplish homeostasis during any perturbance in the lipid composition. Meanwhile, the PAQR-2/IGLR-2 complex works with FLD-1 to balance unsaturated to saturated fatty acids to maintain membrane fluidity. The loss of membrane lipid homeostasis is observed in many human genetic and metabolic disorders. Since C. elegans conserved such genes and pathways, it can be used as a model organism.

Interaction of human phospholipid scramblase 1 with cholesterol via CRAC motif is essential for functional regulation and subcellular localization.

Human phospholipid scramblase 1 (hPLSCR1) possesses a putative cholesterol binding CRAC (cholesterol interaction/recognition amino acid consensus) motif at the C-terminal. The CRAC motif of hPLSCR1 interacts with cholesterol with an energy of interaction -64.39 KJ mol-1. Since palmitoylated hPLSCR1 localizes to the cholesterol-rich lipid rafts, the interaction between hPLSCR1 and raft cholesterol is highly likely. The present study investigated the hPLSCR1-cholesterol interaction in plasma membrane via putative CRAC motif. hPLSCR1 remains at cholesterol-rich lipid rafts as long as they interact. This interaction is inhibited by mutations in the CRAC motif or cholesterol depletion. Thus, CRAC mutants I300D hPLSCR1 and ΔCRAC hPLSCR1 diffused to the cytoplasm and nucleus. Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) dose-dependently reduced cell viability in A549 cells. However, cholesterol depletion released 1.74 ± 0.12 times Ca2+ to the cytosol in A549 cells. Similarly, cholesterol depletion increased intracellular Ca2+ release by 1.81 ± 0.13 and 4.11 ± 0.19 times in RAJI cells expressing hPLSCR1 and ΔCRAC hPLSCR1, respectively. Moreover, the expression of hPLSCR1 and ΔCRAC hPLSCR1 increased apoptosis in RAJI cells by 21 ± 1.5% and 53.50 ± 4.40%, respectively. It was further increased to 43 ± 2.5% and 71.4 ± 1.4% upon cholesterol depletion. The current work links hPLSCR1 expression with cholesterol depletion, intracellular Ca2+ release, and induction of apoptosis.

Cholesterol interaction attenuates scramblase activity of SCRM-1 in the artificial membrane.

Phospholipid (PL) scramblases are single-pass transmembrane protein mediating bidirectional PL translocation. Previously in silico analysis of human PL scramblases, predicted the presence of an uncharacterized cholesterol-binding domain spanning partly in the transmembrane helix as well as in the adjacent extracellular coil. This domain was found to be universally conserved in diverse organisms like Caenorhabditis elegans. In this study, we investigated the saturable cholesterol-binding domain of SCRM-1 using fluorescence sterol binding assay, Stern-Volmer quenching, Förster resonance energy transfer, and CD spectroscopy. We observed high-affinity interaction between cholesterol and SCRM-1. Our results support a previous report, which showed that the cholesterol ordering effect reduced the scramblase activity of hPLSCR1. Considering the presence of a high-affinity binding sequence, we propose that the reduction in activity could partly be due to the cholesterol binding. To validate this, we generated a C-terminal helix (CTH) deletion construct (∆CTH SCRM-1) and a point mutation in the putative cholesterol-binding domain I273D SCRM-1. Deletion construct greatly reduced cholesterol affinity along with loss of scramblase activity. In contrast to this, I273D SCRM-1 retained scrambling activity in proteoliposomes containing ~30 mol% cholesterol but lost sterol binding ability. These results suggest that C-terminal helix is crucial for membrane insertion and in the lipid bilayer the scrambling activity of SCRM-1 is modulated through its interaction with cholesterol.