High-affinity Ca2 Research Articles

High-Affinity Plasma Membrane Ca2+ Channel Cch1 Modulates Adaptation to Sodium Dodecyl Sulfate-Triggered Rise in Cytosolic Ca2+ Concentration in Ogataea parapolymorpha.

The cytosolic calcium concentration ([Ca2+]cyt) in yeast cells is maintained at a low level via the action of different transporters sequestrating these cations in the vacuole. Among them, the vacuolar Ca2+ ATPase Pmc1 crucially contributes to this process. Its inactivation in Ogataea yeasts was shown to cause sodium dodecyl sulfate (SDS) hypersensitivity that can be alleviated by the inactivation of the plasma membrane high-affinity Ca2+ channel Cch1. Here, we show that SDS at low concentrations induces a rapid influx of external Ca2+ into cells, while the plasma membrane remains impermeable for propidium iodide. The inactivation of Pmc1 disturbs efficient adaptation to this activity of SDS. The inactivation of Cch1 partially restores the ability of pmc1 mutant cells to cope with an increased [Ca2+]cyt that correlates with the suppression of SDS hypersensitivity. At the same time, Cch1 is unlikely to be directly involved in SDS-induced Ca2+ influx, since its inactivation does not decrease the amplitude of the rapid [Ca2+]cyt elevation in the pmc1-Δ mutant. The obtained data suggest that the effects of CCH1 inactivation on SDS sensitivity and coping with increased [Ca2+]cyt are related to an additional Cch1 function beyond its direct involvement in Ca2+ transport.

Structural bases for blockade and activation of BK channels by Ba2+ ions.

We studied the impact of Ba2+ ions on the function and structure of large conductance potassium (BK) channels. Ion composition has played a crucial role in the physiological studies of BK channels due to their ability to couple ion composition and membrane voltage signaling. Unlike Ca2+, which activates BK channels through all Regulator of K + Conductance (RCK) domains, Ba2+ has been described as specifically interacting with the RCK2 domain. It has been shown that Ba2+ also blocks potassium permeation by binding to the channel's selectivity filter. The Cryo-EM structure of the Aplysia BK channel in the presence of high concentration Ba2+ here presented (PDBID: 7RJT) revealed that Ba2+ occupies the K+ S3 site in the selectivity filter. Densities attributed to K+ ions were observed at sites S2 and S4. Ba2+ ions were also found bound to the high-affinity Ca2+ binding sites RCK1 and RCK2, which agrees with functional work suggesting that the Ba2+ increases open probability through the Ca2+ bowl site (RCK2). A comparative analysis with a second structure here presented (PDBID: 7RK6), obtained without additional Ba2+, shows localized changes between the RCK1 and RCK2 domains, suggestive of coordinated dynamics between the RCK ion binding sites with possible relevance for the activation/blockade of the channel. The observed densities attributed to Ba2+ at RCK1 and RCK2 sites and the selectivity filter contribute to a deeper understanding of the structural basis for Ba2+'s dual role in BK channel modulation, adding to the existing knowledge in this field.

Calcium Role in Gap Junction Channel Gating: Direct Electrostatic or Calmodulin-Mediated?

The chemical gating of gap junction channels is mediated by cytosolic calcium (Ca2+i) at concentrations ([Ca2+]i) ranging from high nanomolar (nM) to low micromolar (µM) range. Since the proteins of gap junctions, connexins/innexins, lack high-affinity Ca2+-binding sites, most likely gating is mediated by a Ca2+-binding protein, calmodulin (CaM) being the best candidate. Indeed, the role of Ca2+-CaM in gating is well supported by studies that have tested CaM blockers, CaM expression inhibition, testing of CaM mutants, co-localization of CaM and connexins, existence of CaM-binding sites in connexins/innexins, and expression of connexins (Cx) mutants, among others. Based on these data, since 2000, we have published a Ca2+-CaM-cork gating model. Despite convincing evidence for the Ca2+-CaM role in gating, a recent study has proposed an alternative gating model that would involve a direct electrostatic Ca2+-connexin interaction. However, this study, which tested the effect of unphysiologically high [Ca2+]i on the structure of isolated junctions, reported that neither changes in the channel's pore diameter nor connexin conformational changes are present, in spite of exposure of isolated gap junctions to [Ca2+]i as high at the 20 mM. In conclusion, data generated in the past four decades by multiple experimental approaches have clearly demonstrated the direct role of Ca2+-CaM in gap junction channel gating.

Calreticulin-Enigmatic Discovery.

Calreticulin (CRT) is an intrinsically disordered multifunctional protein that plays essential roles intra-and extra-cellularly. The Michalak laboratory has proposed that CRT was initially identified in 1974 by the MacLennan laboratory as the high-affinity Ca2+-binding protein (HACBP) of the sarcoplasmic reticulin (SR). This widely accepted belief has been ingrained in the scientific literature but has never been rigorously tested. In our report, we have undertaken a comprehensive reexamination of this assumption by meticulously examining the majority of published studies that present a proteomic analysis of the SR. These analyses have utilized proteomic analysis of purified SR preparations or purified components of the SR, namely the longitudinal tubules and junctional terminal cisternae. These studies have consistently failed to detect the HACBP or CRT in skeletal muscle SR. We propose that the existence of the HACBP has failed the test of reproducibility and should be retired to the annals of antiquity. Therefore, the scientific dogma that the HACBP and CRT are identical proteins is a non sequitur.

Neurogranin modulates the rate of association between calmodulin and target peptides

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25–1.5 μM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

A note on estimating absolute cytosolic Ca2+ concentration in sensory neurons using a single wavelength Ca2+ indicator.

Ca2+ imaging is frequently used in the investigation of sensory neuronal function and nociception. In vitro imaging of acutely dissociated sensory neurons using membrane-permeant fluorescent Ca2+ indicators remains the most common approach to study Ca2+ signalling in sensory neurons. Fluo4 is a popular choice of single-wavelength indicator due to its brightness, high affinity for Ca2+ and ease of use. However, unlike ratiometric indicators, the emission intensity from single-wavelength indicators can be affected by indicator concentration, optical path length, excitation intensity and detector efficiency. As such, without careful calibration, it can be difficult to draw inferences from differences in the magnitude of Ca2+ transients recorded using Fluo4. Here, we show that a method scarcely used in sensory neurophysiology - first proposed by Maravall and colleagues (2000) - can provide reliable estimates of absolute cytosolic Ca2+ concentration ([Ca2+]cyt) in acutely dissociated sensory neurons using Fluo4. This method is straightforward to implement; is applicable to any high-affinity single-wavelength Ca2+ indicator with a large dynamic range; and provides estimates of [Ca2+]cyt in line with other methods, including ratiometric imaging. Use of this method will improve the granularity of sensory neuron Ca2+ imaging data obtained with Fluo4.

Secretagogin regulates asynchronous and spontaneous glutamate release in hippocampal neurons through interaction with Doc2α

Abstract Synaptic vesicle (SV) exocytosis is orchestrated by protein machineries consisting of the SNARE complex, Ca2+ sensors, and their partners. Secretagogin (SCGN) is a Ca2+-binding protein involved in multiple forms of vesicle secretion. Although SCGN is implicated in multiple neurological disorders, its role in SV exocytosis in neurons remains unknown. Here, using knockout and knockdown techniques, we report that SCGN could regulate the asynchronous and spontaneous forms of excitatory but not inhibitory SV exocytosis in mouse hippocampal neurons. Furthermore, SCGN functioned in glutamate release via directly interacting with Doc2α, a high-affinity Ca2+ sensor specific for asynchronous and spontaneous SV exocytosis. Conversely, the interaction with SCGN is also required for Doc2α to execute its Ca2+ sensor function in SV release. Together, our study revealed that SCGN plays an important role in asynchronous and spontaneous glutamate release through its interaction with Doc2α.

Effect of cysteine mutation at Ca2+ coordinating residues to the autolysis, folding and hydrophobicity of full length and mature Rand protease: molecular dynamics simulation and essential dynamics

Rand protease is a serine protease that shared common characteristics with members of the MEROPS S8 subtilisin family. It is thermostable, highly stable in organic solvent and broad in specificity. Many structures of homologous protein solved by X-ray crystallography and NMR have been deposited to Protein Data Bank (PDB) which allowed this study to rely on structure prediction by deep learning to build three-dimensional (3D) structure of full length and mature Rand protease (flRP and mRP). In silico cysteine mutation to 7 predicted high affinity Ca2+ coordinating residues were introduced, and the mutants were subjected to molecular dynamics simulation to study its effect on flRP and mRP. MD simulation showed a marked increase in flexibility of the pro-peptide segment indicating the impact of single cysteine substitution at high affinity Ca2+ coordinating residues to autolysis of flRP. MD simulation for mRP reaffirmed the role of Ca2+ coordinating sites in providing stability to Rand protease. In addition, these residues also affect the autolysis, folding and hydrophobicity of RP. Essential dynamics observed large contribution of the first few eigenvectors of flRP, mRP and their high affinity Ca2+ coordinating residues mutants to the TMSF values which indicates that these values account for a large portion of the overall atomic fluctuations. These results have given a more comprehensive understanding on the role of cysteine substituted Ca2+ coordinating surface loop to the structure of flRP and mRP which are important in contributing to the structural stability of subtilisin. Communicated by Ramaswamy H. Sarma

Molecular mechanics and failure mechanisms in B. mori Silk Fibroin-hydroxyapatite composite interfaces: Effect of crystal thickness and surface characteristics

Bombyx mori Silk Fibroin-hydroxyapatite (B. mori SF-HA) bio-nanocomposite is a prospective biomaterial for tissue engineered graft for bone repair. Here, B. mori SF is primarily a soft and tough organic phase, and HA is a hard and stiff mineral phase. In biomaterial design, an understanding about the nanoscale mechanics of SF-HA interface, such as interfacial interaction and interface debonding mechanisms between the two phases is essential for obtaining required functionality. To investigate such nanoscale behavior, molecular dynamics method is a preferred approach. Present study focuses on understanding of the interface debonding mechanisms at SF-HA interface in B. mori SF-HA bio-nanocomposite at nanometer length scale. For this purpose, nanoscale atomistic models of SF-HA interface are also developed based on the HA crystal size and HA surface type (Ca2+ dominated and OH− dominated) in contact with SF. Mechanical behavior analysis of these SF-HA interface models under pull-out type test were performed using Molecular Dynamics (MD) simulations. Surface pull-off strength values in the range of 0.4–0.8 GPa were obtained for SF-HA interface models, for different HA crystal thicknesses, wherein, the pull-off strength values are found to increase with increase in HA thicknesses. Analyses show that deformation mechanisms in SF-HA interface deformation, is a combination of shear deformation in SF phase followed by disintegration of SF phase from HA block. Furthermore, higher rupture force values were obtained for SF-HA interface with Ca2+ dominated HA surface in contact with SF phase, indicating that SF protein has a higher affinity for Ca2+ dominated surface of HA phase. Current work contributes in developing an understanding of mechanistic interactions between organic and inorganic phases in B. mori SF-HA composite nanostructure.

Plasma membrane Ca2+ pump isoform 4 function in cell migration and cancer metastasis.

The Ca2+ ion is a universal second messenger involved in many vital physiological functions including cell migration and development. To fulfil these tasks the cytosolic Ca2+ concentration is tightly controlled, and this involves an intricate functional balance between a variety of channels and pumps of the Ca2+ signalling machinery. Among these proteins, plasma membrane Ca2+ ATPases (PMCAs) represent the major high-affinity Ca2+ extrusion systems in the cell membrane that are effective in maintaining free Ca2+ concentration at exceedingly low cytosolic levels, which is essential for normal cell function. An imbalance in Ca2+ signalling can have pathogenic consequences including cancer and metastasis. Recent studies have highlighted the role of PMCAs in cancer progression and have shown that a particular variant, PMCA4b, is downregulated in certain cancer types, causing delayed attenuation of the Ca2+ signal. It has also been shown that loss of PMCA4b leads to increased migration and metastasis of melanoma and gastric cancer cells. In contrast, an increased PMCA4 expression has been reported in pancreatic ductal adenocarcinoma that coincided with increased cell migration and shorter patient survival, suggesting distinct roles of PMCA4b in various tumour types and/or different stages of tumour development. The recently discovered interaction of PMCAs with basigin, an extracellular matrix metalloproteinase inducer, may provide further insights into our understanding of the specific roles of PMCA4b in tumour progression and cancer metastasis.

Ca2+-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins

Ca2+/calmodulin (Ca2+/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca2+/CaM is not fully understood. Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50-100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.

Structural bases for blockade and activation of BK channels by Ba2+ ions.

Calcium ions increase the open probability of the large conductance potassium (BK) channels by binding to high affinity sites within the large cytosolic Ca2+ sensing structure called the gating ring. This Ca2+ sensing structure is formed by eight Regulator of K+ Conductance (RCK) domains, two from each subunit of the channel, termed RCK1 and RCK2. Other divalent species, like Ba2+ ions, are also able to activate BK channels by interacting with the Ca2+ sensor. Unlike Ca2+ that activate BK channel through all RCK domains, Ba2+ does it selectively through the RCK2 domain. In addition to activation, Ba2+ can also block K+ permeation by binding to the selectivity filter of BK channels. Here we present and discuss Cryo-EM structures of BK channels with Ba2+. Structurally, blockade arises from one Ba2+ occupying the traditional K+ site S3 in the selectivity filter of the channel. In addition, electronic densities attributed to K+ ions are detected at K+ sites S2 and S4. In the gating ring, eight Ba2+ ions are bound to the corresponding high affinity Ca2+ binding sites. Thereby, the lack of functional significance of the RCK1 on the activation by Ba2+ should arise because the architectures of the gating rings in our structures represent intermediate transitions between the close and the open configurations. Finally, these Ba2+ structures reveal an intricate series of concerted changes of the RCK 1 and RCK2 from the same subunit, suggesting coordinated intra-subunit dynamics.

Neurotransmitter release progressively desynchronizes in induced human neurons during synapse maturation and aging.

Rapid release of neurotransmitters in synchrony with action potentials is considered a key hardwired property of synapses. Here, in glutamatergic synapses formed between induced human neurons, we show that action potential-dependent neurotransmitter release becomes progressively desynchronized as synapses mature and age. In this solely excitatory network, the emergence of NMDAR-mediated transmission elicits endoplasmic reticulum (ER) stress leading to downregulation of key presynaptic molecules, synaptotagmin-1 and cysteine string protein α, that synchronize neurotransmitter release. The emergence of asynchronous release with neuronal maturity and subsequent aging is maintained by the high-affinity Ca2+ sensor synaptotagmin-7 and suppressed by the introduction of GABAergic transmission into the network, inhibition of NMDARs, and ER stress. These results suggest that long-term disruption of excitation-inhibition balance affects the synchrony of excitatory neurotransmission in human synapses.

Calcium-dependent modulation of BKCa channel activity induced by plasmonic gold nanoparticles in pulmonary artery smooth muscle cells and hippocampal neurons.

Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon-induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5nm gold nanoparticles on calcium-dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. Outward potassium currents were recorded using patch-clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo-3 and laser confocal microscope. In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large-conductance Ca2+ -activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles-induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium-permeable ion channels, which leads to a change in intracellular calcium level.

Dark-coloured Mn-rich overgrowths in an elbaitic tourmaline crystal from the Rosina pegmatite, San Piero in Campo, Elba Island, Italy: witness of late-stage opening of the geochemical system

AbstractMulticoloured tourmalines from Elba Island, commonly display dark-coloured terminations due to incorporation of Fe, and also occasionally Mn. The mechanisms which led to the availability of these elements in the late-stage residual fluids are not yet completely understood. For this purpose, we investigated a representative tourmaline crystal found naturally in two fragments within a wide miarolitic cavity in the Rosina pegmatite (San Piero in Campo, Elba Island, Italy), and characterised by late-stage dark-coloured overgrowths. Microstructural and paragenetic observations, together with compositional and spectroscopic data (electron microprobe and optical absorption spectroscopy), provide evidence which shows that the formation of the dark-coloured Mn-rich overgrowths are the result of a pocket rupture. This event caused alteration of the cavity-coating spessartine garnet by highly-reactive late-stage cavity fluids by leaching processes, with the subsequent release of Mn to the residual fluids. We argue that the two fragments were originally a single crystal, which underwent natural breakage followed by the simultaneous growth of Mn-rich dark terminations at both breakage surfaces. This conclusion supports the evidence for a pocket rupture event, responsible for both the shattering of the tourmaline crystal and the compositional variation of the cavity-fluids related to the availability of Mn, which was incorporated by the tourmaline crystals. Additionally, a comparison of the dark overgrowths formed at the analogous and the antilogous poles, provides information on tourmaline crystallisation at the two different poles. The antilogous pole is characterised by a higher affinity for Ca, F and Ti, and a selective uptake of Mn2+, even in the presence of a considerable amount of Mn3+ in the system. This uneven uptake of Mn ions resulted in the yellow–orange colouration of the antilogous overgrowth (Mn2+ dependent) rather than the purple-reddish colour of the analogous overgrowths (Mn3+ dependent).

Investigation on Controlling Therapy of Bone Skeletal and Marrow Cancer: A Biophysical Chemistry and Molecular Dynamic Study of Bisphosphonates Interaction with Bone Structures

For more than four decades, the bisphosphonates family has been applied for osteoporosis and skeletal metastasis therapy. These drugs decrease the viability of cancer cells that are guided through the HER group of receptor tyrosine kinases. We discussed that bisphosphonates straightly bind to and inhibit HER kinases. In this study for docking a nitrogen-containing bisphosphonate with human FPPS and a few other targets, the iGEMDOCK docking software has been used. Nitrogen-containing bisphosphonates (NBPs) are mostly applied for bone treatment and also for the loss of skeletal disorders. The adsorption, retention, diffusion, and release of (NBPs) in bone are controlled by their affinities to such mineral compounds. Bisphosphonates have a high affinity for Ca2+ and therefore attack bone minerals, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. Nitrogen-containing bisphosphonates (NBPs), including Alendronate, Zolendronate, Risedronic, Ibandronate, and Pamidronate, are functionalized as effective inhibitors of bone resorption diseases. It targets FPPS (osteoclast farnesyl pyrophosphate synthase) to inhibit protein prenylation. Generally, the strong interaction sequence is as follows Alendronate > Risedronic > Pamidronate > Zolendronate > Ibandronate, and this was because of strong electrostatic interactions between amine groups and phosphate ions.

Fast resupply of synaptic vesicles requires synaptotagmin-3.

Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Ca2+ signals by an as-yet unidentified high-affinity Ca2+ sensor1,2. Here we identify synaptotagmin-3 (SYT3)3,4 as that presynaptic high-affinity Ca2+ sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Ca2+. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref. 5). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Ca2+-dependent vesicle docking.

The tumor neomatrix component fibrillin-1 (FBN1) is a druggable oncologically relevant target: Proof-of-principle in vitro using an FDA-approved medicine as pioneer inhibitor of protein hydroxylation (274)

<b>Objectives:</b> Fibrillins <i>(FBN1-4)</i> determine the structure and function of the extracellular matrix. In malignancies, they drive the cytokine-loaded microenvironment and neoangiogenesis that promote tumor growth. In high-grade serous ovarian cancer (HGSOC), <i>FBN1</i> ranks among the top five genes with robust prognostic signature for survival [Ann. Oncol 31, 1240-50 (2020)]. Fibrillin-1 displays ≈60 globules aligned as ‘string-of-pearls,' ≈45 being epidermal growth factor domains (EGFDs); ≈40 EGFDs are hydroxylated at Asp/Asn residues by AspH, a 2-oxoacid utilizing dioxygenase (2OUD). AspH forms a high-affinity Ca²⁺ binding site in-between adjacent EGFDs, stabilizing the elastic alignment of ‘pearls' within fibrillin. We hypothesized that AspH inhibition causes misfolding of fibrillin due to failed Ca²⁺ binding, thus flawed alignment of EGFDs and intracellular retention of this intentionally defective fibrillin, similar to intracellular retention of genetically defective fibrillin in Marfan. We aimed to Identify a pioneer fibrillin-1 inhibitor that disrupts fibrillin synthesis preferentially in cancer cells, blocks any fibrillin contribution to the biology of solid tumors and has potential for clinical testing in ovarian serous and uterine serous cancers (USC). <b>Methods:</b> Crystal structure-guided modeling; cultured cell lines with high genomic fidelity to tissue and validation in drug discovery (MRC5 [normal fibroblasts, NF], KURAMOCHI [HGSOC], ARK1 [USC]); RNA-seq analysis; multi-channel flow cytometry. <b>Results:</b> To identify a medically actionable AspH suppressor for testing our hypothesis, we measured the inhibition-pertinent 3D parameters of the HAG mechanism, known to resolve 2OUD catalysis at the scale of atomic orbitals (https://www.researchgate.net/publication/7621997). We applied this HAG parameter grid to the library of FDA-approved drugs and selected deferiprone (DEF) as a candidate AspH suppressor. Globally, thousands of thalassemic children use DEF for a lifetime to alleviate transfusional iron overload. <i>In silico,</i> we found DEF accesses the catalytic pocket of AspH and blocks the two coordination sites at its iron atom that are essential for utilizing molecular oxygen during EGFD hydroxylations in fibrillins. <i>In vitro,</i> ARK1 transcribes <i>FBN1, FBN2,</i> and <i>FBN3.</i> MRC5, KURAMOCHI, and ARK1 produce fibrillin-1, detected by the FITC-labeled monoclonal 11C1.3, specific for a unique epitope within residues 723-909 of fibrillin-1 (exons 18-20 of <i>FBN1).</i> Employed at clinically relevant concentrations (≤ 150 µM), DEF treatment caused dose-dependent intracellular accumulation of 11C1.3-reactive fibrillin-1 material in all cell lines, exceeding untreated controls by up to five-fold after 48 hrs (p≤0.05). After 96 hours on DEF, such accumulation resolved in non-malignant MRC5 but persisted in malignant KURAMOCHI (p=0.04) and increased even further in malignant ARK1 (p≤0.02). Measured simultaneously, cleaved <i>PARP-1</i>, a signature of apoptotic death, was downregulated in DEF-treated MRC5 yet was elevated up to four-fold over controls in DEF-treated KURAMOCHI (p<0.03) and DEF-treated ARK1 (p<0.006). These increases correlated directly with the accumulation of 11C1.3-reactive material (Pearson's r: 0.97 in Kuramochi, 0.99 in ARK1) <b>Conclusions:</b> DEF causes intracellular accumulation of fibrillin-1, mis- or unfolded by deficient hydroxylation, and triggers apoptotic death in HGSOC and USC, but not in NF cells. Our results encourage a DEF pilot trial in HGSOC/USC patients.

Mn2+ modulates the production of mycophenolic acid in Penicillium brevicompactum NRRL864 via reactive oxygen species signaling and the investigation of pb-pho

Although Penicillium brevicompactum is a widely used commercial strain for the manufacture of mycophenolic acid, there are few findings of Ca2+, Mn2+, and reactive oxygen species (ROS) interaction. Metal ions play a crucial role in physiological metabolism. Calcium, as the important second messenger, influences fungus growth, virulence, and stress responses. The concentration of cytosolic Ca2+ was influenced by the Mn2+, which demonstrated the crosstalk between calcium and manganese. In the previous study, the crosstalk between calcium and ROS has been discovered and verified, which modified the secondary metabolism and enhanced the yield of MPA (Mycophenolic Acid). A higher concentration of Mn2+ in the fermentation broth causes an increase in cytoplasmic Ca2+ and ROS (Reactive Oxygen Species), enhancing the yield of MPA by about 20 % higher and disclosing the cascade regulation with the Mn2+, Ca2+, and ROS. To be more specific, the intracellular concentration of ROS at 6 mM Mn2+ is about 1.5 times higher than that at 0.6 mM. Furthermore, we identify an Mn2+ transport protein, designated as Pb–PHO, which shows 71.2 % identity to the inorganic phosphate transporter PHO84 (Q0CBJ6) from Aspergillus terreus. At the same time, the △Pb-pho exhibits damage to the cell wall integrity, while the OE-pho displays a more normal phenotype at high osmotic stress. The high-affinity Ca2+ channel, Pb–CCH, is examined via knockdown to demonstrate the crosstalk between Mn2+ and Ca2+. The results show that the addition of Mn2+ remits the negative influence of pb-cch knockdown and the addition of Ca2+ remits the negative influence of pb-pho knockdown, demonstrating the relationship between cytoplasmic Mn2+ and Ca2+. Taken together, our results demonstrate the mechanism of a manganese-induced cascade of manganese-calcium-ROS and reveal a signal pathway-relative method to illustrate the manganese-induced increase of MPA production in Penicillium brevicompactum. Furthermore, we discover and identify an Mn2+ transport protein, Pb–PHO, which is subcellular localized at the plasma membrane and proved to affect the cell wall integrity.

Synaptotagmins 1 and 7 Play Complementary Roles in Somatodendritic Dopamine Release.

The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca2+ concentration ([Ca2+]o). Here we tested the hypothesis that the high-affinity Ca2+ sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca2+ dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca2+ dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca2+]o in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca2+]o, pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca2+]o than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons.SIGNIFICANCE STATEMENT The respective Ca2+ dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca2+ concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca2+ sensor, underlies phasic somatodendritic DA release and its Ca2+ sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca2+ sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca2+ sensors subserving different aspects of somatodendritic DA release processes.