In addition to its function of innate immunity against invading pathogens, neutrophil extracellular traps (NETs) promote thrombosis, autoimmune disease, and cancer metastasis; therefore, unnecessary exposure to the triggers of infection-independent NET generation should be avoided. We herein show that inhibition of forward-mode Na⁺/Ca²⁺ exchange by amiloride analogs, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N-Methyl-N-isobutyl)amiloride (MIA), triggers NETotic cell death independently of infectious stimuli. Isolated human neutrophils treated with EIPA and MIA undergo NETotic cell death by an increase of intracellular Ca²⁺ following activation of NADPH oxidase and the resultant upregulation of intracellular ROS. EIPA- and MIA-mediated intracellular Ca²⁺ increase is attributed to the competitive binding of EIPA and MIA against Na⁺ to Na⁺/Ca²⁺ exchanger 1 (NCX1). These results demonstrate a new mechanism of infection-independent NET generation and implicate NCX1 as a physiologic regulator of intracellular calcium balance and NETotic cell death.

KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca²⁺-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca²⁺-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca²⁺-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)–based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca²⁺ influx by extracellular ligands, KRAS4b reversibly translocated in a Ca²⁺-CaM–dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca²⁺ signaling pathways.

Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Studies on GnRH have demonstrated that GnRH has both stimulatory and inhibitory effects on cell proliferation depending on the cell type; however, the mechanisms of these effects remain to be elucidated. Against this background we used four human cell lines, TSU-Pr1, Jurkat, HHUA and DU145, and newly found that GnRH increased or decreased the colony-formation depending on the cell line. Moreover, we demonstrated that the stimulatory and inhibitory effects of GnRH exhibit distinct ligand selectivities. In order to investigate the molecular basis of these phenomena, analyses of the expression of human GnRH receptors were performed and, moreover, the effects of GnRH were analyzed under conditions in which human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH...

GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Progress in studies on GnRH demonstrated that GnRH has both inhibitory and stimulatory effects on cell proliferation depending on the cell type, and the mechanisms of these effects have been intensively studied. However, even human GnRH receptors which mediate GnRH stimulation have not been completely identified. In the present study, we showed that the inhibitory and stimulatory effects of GnRH on colony-formation using four cell lines and have demonstrated that the inhibitory and stimulatory effects of GnRH exhibit distinctly different patterns of ligand sensitivity. This result strongly suggests that the two opposite effects of GnRH occur via different types of GnRH receptors, however expressional analyses of human GnRH receptors did not exhibit the significantly different pattern between negatively and positively responding cell lines. Then, in order to identify the GnRH receptors involved in the two opposite effects, effects of GnRH were analysed under the conditions that human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor mediates GnRH stimulation and its splice variant determines the direction of the response to GnRH. These results are the first clear evidence for the functionality of human type II GnRH receptor. Therefore our novel findings are quite noticeable and will greatly contribute to the studies on the mechanisms of the effects of GnRH on cell proliferation in the future.

The ordered assembly of α-synuclein protein encoded by SNCA into filaments characterizes neurodegenerative synucleinopathies. Lewy body disease (LBD) shows predominantly neuronal and multiple system atrophy (MSA), predominantly oligodendrocytic α-synuclein pathology affecting subcortical brain structures. Based on cryo-electron microscopy, it was reported that the structures of α-synuclein filaments from LBD differ from MSA and juvenile-onset synucleinopathy (JOS). The rare atypical MSA subtype shows abundant neuronal argyrophilic α-synuclein inclusions in the limbic system. Current concepts indicate that disease entities are characterized by unique protofilament folds. Here we demonstrate that α-synuclein can form a Lewy-MSA hybrid fold, leading to the atypical histopathological form of MSA. Distinct biochemical characteristics of α-synuclein, as demonstrated by protease-sensitivity digestion assay, seed amplification assays (SAAs), and conformational stability assays (CSA), are also linked to cytopathological differences. We expand the current structure-based classification of α−synucleinopathies and propose that cell-specific protein pathologies can be associated with distinct filament folds.