Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10–30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

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Liaison between Myristoylation and Cryptic EF-Hand Motif Confers Ca2+ Sensitivity to Neuronal Calcium Sensor-1

Biochemistry ◽

10.1021/bi501134g ◽

2015 ◽

Vol 54 (4) ◽

pp. 1111-1122 ◽

Cited By ~ 5

Author(s):

Vangipurapu Rajanikanth ◽

Anand Kumar Sharma ◽

Meduri Rajyalakshmi ◽

Kousik Chandra ◽

Kandala V. R. Chary ◽

...

Keyword(s):

Calcium Sensor ◽

Neuronal Calcium Sensor ◽

Ef Hand

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Oxidative stress is involved in Patulin induced apoptosis in HEK293 cells

Toxicon ◽

10.1016/j.toxicon.2014.12.002 ◽

2015 ◽

Vol 94 ◽

pp. 1-7 ◽

Cited By ~ 36

Author(s):

Baigang Zhang ◽

Xiaoli Peng ◽

Guanghui Li ◽

Yunfeng Xu ◽

Xiaodong Xia ◽

...

Keyword(s):

Oxidative Stress ◽

Hek293 Cells ◽

Induced Apoptosis

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Single-Molecule Folding Mechanism of an EF-Hand Neuronal Calcium Sensor

Structure ◽

10.1016/j.str.2013.07.022 ◽

2013 ◽

Vol 21 (10) ◽

pp. 1812-1821 ◽

Cited By ~ 18

Author(s):

Pétur O. Heidarsson ◽

Mariela R. Otazo ◽

Luca Bellucci ◽

Alessandro Mossa ◽

Alberto Imparato ◽

...

Keyword(s):

Single Molecule ◽

Calcium Sensor ◽

Neuronal Calcium Sensor ◽

Folding Mechanism ◽

Ef Hand

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Lovastatin Alleviates α-Synuclein Aggregation and Phosphorylation in Cellular Models of Synucleinopathy

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.682320 ◽

2021 ◽

Vol 14 ◽

Author(s):

Lijun Dai ◽

Jiannan Wang ◽

Mingyang He ◽

Min Xiong ◽

Ye Tian ◽

...

Keyword(s):

Oxidative Stress ◽

Neurodegenerative Diseases ◽

Lower Risk ◽

Clinical Evidence ◽

Hek293 Cells ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cellular Models ◽

Potential Value

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Pathologically, it is characterized by the aberrant aggregation of α-synuclein (α-syn) in neurons. Clinical evidence shows that patients with hypercholesterolemia are more likely to get PD, while lovastatin users have a lower risk of suffering from it. In this study, we investigated the effects of lovastatin on the aggregation and phosphorylation of α-syn in vitro. Our results demonstrate that α-syn preformed fibrils induce the phosphorylation and aggregation of α-syn in HEK293 cells stably transfected with α-syn-GFP and SH-SY5Y cells as well, which could be attenuated by in a concentration-dependent manner. Besides, lovastatin inhibited oxidative stress, histone acetylation, and the activation of casein kinase 2 (CK2). Collectively, lovastatin alleviates α-syn aggregation and phosphorylation in cellular models of synucleinopathy, indicating its potential value of being adopted in the management of PD.

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Regulatory and Structural EF-Hand Motifs of Neuronal Calcium Sensor-1: Mg2+ Modulates Ca2+ Binding, Ca2+-Induced Conformational Changes, and Equilibrium Unfolding Transitions

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.12.033 ◽

2008 ◽

Vol 376 (4) ◽

pp. 1100-1115 ◽

Cited By ~ 51

Author(s):

Penmatsa Aravind ◽

Kousik Chandra ◽

Pasham Parameshwar Reddy ◽

Andreas Jeromin ◽

K.V.R. Chary ◽

...

Keyword(s):

Conformational Changes ◽

Calcium Sensor ◽

Neuronal Calcium Sensor ◽

Ef Hand ◽

Equilibrium Unfolding

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Cadmium directly induced mitochondrial dysfunction of human embryonic kidney cells

Human & Experimental Toxicology ◽

10.1177/0960327110384286 ◽

2010 ◽

Vol 30 (8) ◽

pp. 920-929 ◽

Cited By ~ 24

Author(s):

WP Mao ◽

NN Zhang ◽

FY Zhou ◽

WX Li ◽

HY Liu ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dysfunction ◽

Hek293 Cells ◽

Human Embryonic Kidney Cells ◽

Stress Markers ◽

Mitochondria Pathway ◽

Induced Apoptosis ◽

And Function ◽

Induced Changes ◽

First Time

Cadmium (Cd) is the major component of polluted environment, which has numerous undesirable effects on health. Cd could induce apoptosis of HEK293 cells, and the mitochondria may play a key role. However, the mode of action is unclear. In the present study, we aimed to evaluate the ability of the Cd to induce dysfunction of mitochondria. We examined the effect of cadmium chloride (1, 5 and 10 μM) on mitochondrial membrane permeability and potential as well as oxidative stress markers in mitochondria isolated from HEK293 cells. We found that Cd could directly increase in permeability and decrease in membrane potential of mitochondria, even resulted in mitochondrial swelling, and that Cd could inhibit the activities of ATPase, lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), enhanced the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO). On the whole, the results show that Cd can directly lead to mitochondrial dysfunction of HEK293 cells, including increased permeability, inhibiting respiration and evoking oxidative stress. Thus, for the first time, this paper makes an overall analysis of Cd-induced changes of structure and function of isolated mitochondria. Our findings may also have general implications in Cd-induced apoptosis by mitochondria pathway.

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Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide

Biochemical Journal ◽

10.1042/bj20030376 ◽

2003 ◽

Vol 375 (1) ◽

pp. 87-97 ◽

Cited By ~ 7

Author(s):

Miki OKADA ◽

Daisuke TAKEZAWA ◽

Shuji TACHIBANAKI ◽

Satoru KAWAMURA ◽

Hiroshi TOKUMITSU ◽

...

Keyword(s):

Protein Family ◽

Kinase Assay ◽

Regulatory Function ◽

Dependent Manner ◽

Calcium Sensor ◽

Neuronal Calcium Sensor ◽

Ef Hand ◽

Ic50 Values ◽

Sensor Proteins ◽

Calcium Sensor Proteins

The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK (Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 μM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 μM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked KATP channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists.

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