Mechanism of Zn2+ and Ca2+ Binding to Human S100A1

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 μm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

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Integrated transcriptomic and proteomic analysis reveals the complex molecular mechanisms underlying stone cell formation in Korla pear

Scientific Reports ◽

10.1038/s41598-021-87262-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aisajan Mamat ◽

Kuerban Tusong ◽

Juan Xu ◽

Peng Yan ◽

Chuang Mei ◽

...

Keyword(s):

Cell Differentiation ◽

Proteomic Analysis ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Cell Formation ◽

Parenchyma Cell ◽

Lignin Biosynthesis ◽

Cell Content ◽

Stone Cell ◽

Fruit Samples

AbstractKorla pear (Pyrus sinkiangensis Yü) is a landrace selected from a hybrid pear species in the Xinjiang Autonomous Region in China. In recent years, pericarp roughening has been one of the major factors that adversely affects fruit quality. Compared with regular fruits, rough-skin fruits have a greater stone cell content. Stone cells compose sclerenchyma tissue that is formed by secondary thickening of parenchyma cell walls. In this work, we determined the main components of stone cells by isolating them from the pulp of rough-skin fruits at the ripening stage. Stone cell staining and apoptosis detection were then performed on fruit samples that were collected at three different developmental stages (20, 50 and 80 days after flowering (DAF)) representing the prime, late and stationary stages of stone cell differentiation, respectively. The same batches of samples were used for parallel transcriptomic and proteomic analysis to identify candidate genes and proteins that are related to SCW biogenesis in Korla pear fruits. The results showed that stone cells are mainly composed of cellulose (52%), hemicellulose (23%), lignin (20%) and a small amount of polysaccharides (3%). The periods of stone cell differentiation and cell apoptosis were synchronous and primarily occurred from 0 to 50 DAF. The stone cell components increased abundantly at 20 DAF but then decreased gradually. A total of 24,268 differentially expressed genes (DEGs) and 1011 differentially accumulated proteins (DAPs) were identified from the transcriptomic and proteomic data, respectively. We screened the DEGs and DAPs that were enriched in SCW-related pathways, including those associated with lignin biosynthesis (94 DEGs and 31 DAPs), cellulose and xylan biosynthesis (46 DEGs and 18 DAPs), S-adenosylmethionine (SAM) metabolic processes (10 DEGs and 3 DAPs), apoplastic ROS production (16 DEGs and 2 DAPs), and cell death (14 DEGs and 6 DAPs). Among the identified DEGs and DAPs, 63 significantly changed at both the transcript and protein levels during the experimental periods. In addition, the majority of these identified genes and proteins were expressed the most at the prime stage of stone cell differentiation, but their levels gradually decreased at the later stages.

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The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

Biomolecules ◽

10.3390/biom11010066 ◽

2021 ◽

Vol 11 (1) ◽

pp. 66

Author(s):

Alisa A. Vologzhannikova ◽

Polina A. Khorn ◽

Marina P. Shevelyova ◽

Alexei S. Kazakov ◽

Victor I. Emelyanenko ◽

...

Keyword(s):

Solvent Accessibility ◽

Sensory Function ◽

Hydrophobic Residues ◽

Metal Free ◽

Helix Loop Helix ◽

Redox Sensor ◽

Ef Hand ◽

Functional Consequences ◽

Ef Hands ◽

Ph Range

Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca2+-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca2+-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pKa of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca2+-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg2+-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca2+/Mg2+ affinity of the EF site, and accelerates Ca2+ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca2+ affinity.

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Tripodal, Squaramide-Based Ion Pair Receptor for Effective Extraction of Sulfate Salt

Molecules ◽

10.3390/molecules26092751 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2751

Author(s):

Damian Jagleniec ◽

Marcin Wilczek ◽

Jan Romański

Keyword(s):

Ion Pairs ◽

Binding Mode ◽

Selective Extraction ◽

Ion Pair ◽

Hofmeister Series ◽

Sulfate Salt ◽

Lower Affinity ◽

High Affinity ◽

Binding Domains ◽

Hydrated Sulfate

Combining three features—the high affinity of squaramides toward anions, cooperation in ion pair binding and preorganization of the binding domains in the tripodal platform—led to the effective receptor 2. The lack of at least one of these key elements in the structures of reference receptors 3 and 4 caused a lower affinity towards ion pairs. Receptor 2 was found to form an intramolecular network in wet chloroform, which changed into inorganic–organic associates after contact with ions and allowed salts to be extracted from an aqueous to an organic phase. The disparity in the binding mode of 2 with sulfates and with other monovalent anions led to the selective extraction of extremely hydrated sulfate anions in the presence of more lipophilic salts, thus overcoming the Hofmeister series.

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When the Balance Tips: Dysregulation of Mitochondrial Dynamics as a Culprit in Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22094617 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4617

Author(s):

Styliana Kyriakoudi ◽

Anthi Drousiotou ◽

Petros P. Petrou

Keyword(s):

Insulin Resistance ◽

Mitochondrial Function ◽

Human Disease ◽

Molecular Mechanisms ◽

Mitochondrial Dynamics ◽

Mitochondrial Fusion ◽

Mitochondrial Morphology ◽

Disease Development ◽

Pathological Conditions ◽

Wide Range

Mitochondria are dynamic organelles, the morphology of which is tightly linked to their functions. The interplay between the coordinated events of fusion and fission that are collectively described as mitochondrial dynamics regulates mitochondrial morphology and adjusts mitochondrial function. Over the last few years, accruing evidence established a connection between dysregulated mitochondrial dynamics and disease development and progression. Defects in key components of the machinery mediating mitochondrial fusion and fission have been linked to a wide range of pathological conditions, such as insulin resistance and obesity, neurodegenerative diseases and cancer. Here, we provide an update on the molecular mechanisms promoting mitochondrial fusion and fission in mammals and discuss the emerging association of disturbed mitochondrial dynamics with human disease.

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BMP signaling alters aquaporin-4 expression in the mouse cerebral cortex

Scientific Reports ◽

10.1038/s41598-021-89997-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kazuya Morita ◽

Naoyuki Matsumoto ◽

Kengo Saito ◽

Toshihide Hamabe-Horiike ◽

Keishi Mizuguchi ◽

...

Keyword(s):

Cerebral Cortex ◽

Molecular Mechanisms ◽

Water Movement ◽

Water Channel ◽

Bmp Signaling ◽

Aquaporin 4 ◽

Mammalian Brain ◽

Physiological Conditions ◽

Pathological Conditions ◽

Mouse Cerebral Cortex

AbstractAquaporin-4 (AQP4) is a predominant water channel expressed in astrocytes in the mammalian brain. AQP4 is crucial for the regulation of homeostatic water movement across the blood–brain barrier (BBB). Although the molecular mechanisms regulating AQP4 levels in the cerebral cortex under pathological conditions have been intensively investigated, those under normal physiological conditions are not fully understood. Here we demonstrate that AQP4 is selectively expressed in astrocytes in the mouse cerebral cortex during development. BMP signaling was preferentially activated in AQP4-positive astrocytes. Furthermore, activation of BMP signaling by in utero electroporation markedly increased AQP4 levels in the cerebral cortex, and inhibition of BMP signaling strongly suppressed them. These results indicate that BMP signaling alters AQP4 levels in the mouse cerebral cortex during development.

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An in vivo model allowing continuous observation of human vascular formation in the same animal over time

Scientific Reports ◽

10.1038/s41598-020-80497-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yohei Tsukada ◽

Fumitaka Muramatsu ◽

Yumiko Hayashi ◽

Chiaki Inagaki ◽

Hang Su ◽

...

Keyword(s):

Blood Vessel ◽

Blood Vessels ◽

Human Blood ◽

Molecular Mechanisms ◽

Human Tumor ◽

Continuous Observation ◽

In Vivo Models ◽

Cranial Window ◽

Pathological Conditions

AbstractAngiogenesis contributes to numerous pathological conditions. Understanding the molecular mechanisms of angiogenesis will offer new therapeutic opportunities. Several experimental in vivo models that better represent the pathological conditions have been generated for this purpose in mice, but it is difficult to translate results from mouse to human blood vessels. To understand human vascular biology and translate findings into human research, we need human blood vessel models to replicate human vascular physiology. Here, we show that human tumor tissue transplantation into a cranial window enables engraftment of human blood vessels in mice. An in vivo imaging technique using two-photon microscopy allows continuous observation of human blood vessels until at least 49 days after tumor transplantation. These human blood vessels make connections with mouse blood vessels as shown by the finding that lectin injected into the mouse tail vein reaches the human blood vessels. Finally, this model revealed that formation and/or maintenance of human blood vessels depends on VEGFR2 signaling. This approach represents a useful tool to study molecular mechanisms of human blood vessel formation and to test effects of drugs that target human blood vessels in vivo to show proof of concept in a preclinical model.

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Pharmacological Effects and Potential Clinical Usefulness of Polyphenols in Benign Prostatic Hyperplasia

Molecules ◽

10.3390/molecules26020450 ◽

2021 ◽

Vol 26 (2) ◽

pp. 450

Author(s):

Kensuke Mitsunari ◽

Yasuyoshi Miyata ◽

Tomohiro Matsuo ◽

Yuta Mukae ◽

Asato Otsubo ◽

...

Keyword(s):

Benign Prostatic Hyperplasia ◽

Molecular Mechanisms ◽

Prostatic Hyperplasia ◽

Pathological Conditions ◽

Urinary Tract Symptoms ◽

Anti Inflammatory ◽

Lower Urinary

Benign prostatic hyperplasia (BPH) is arguably the most common benign disease among men. This disease is often associated with lower urinary tract symptoms (LUTS) in men and significantly decreases the quality of life. Polyphenol consumption reportedly plays an important role in the prevention of many diseases, including BPH. In recent years, in addition to disease prevention, many studies have reported the efficacy and safety of polyphenol treatment against various pathological conditions in vivo and in vitro. Furthermore, numerous studies have also revealed the molecular mechanisms of the antioxidant and anti-inflammatory effects of polyphenols. We believe that an improved understanding of the detailed pharmacological roles of polyphenol-induced activities at a molecular level is important for the prevention and treatment of BPH. Polyphenols are composed of many members, and their biological roles differ. In this review, we first provide information regarding the pathological roles of oxidative stress and inflammation in BPH. Next, the antioxidant and anti-inflammatory effects of polyphenols, including those of flavonoids and non-flavonoids, are discussed. Finally, we talk about the results and limitations of previous clinical trials that have used polyphenols in BPH, with particular focus on their molecular mechanisms of action.

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Transmembrane-truncated αvβ3 integrin retains high affinity for ligand binding: evidence for an ‘inside-out’ suppressor?

Biochemical Journal ◽

10.1042/bj3300861 ◽

1998 ◽

Vol 330 (2) ◽

pp. 861-869 ◽

Cited By ~ 59

Author(s):

J. Raj MEHTA ◽

Beate DIEFENBACH ◽

Alex BROWN ◽

Eilish CULLEN ◽

Alfred JONCZYK ◽

...

Keyword(s):

Ligand Binding ◽

Wound Repair ◽

Molecular Mechanisms ◽

Full Length ◽

Αvβ3 Integrin ◽

Cytoplasmic Domains ◽

Bivalent Cation ◽

High Affinity ◽

Cellular Environment

The molecular mechanisms of αvβ3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human αvβ3 (r-αvβ3) and compared the activation state of these with αvβ3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-αvβ3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental αvβ3 and the receptor in situ on the cell surface. r-αvβ3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-αvβ3. r-αvβ3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native αvβ3. On M21-L4 melanoma cells, αvβ3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated αIIbβ3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified αvβ3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of αIIbβ3 in situ, intracellular controls lower the affinity of αvβ3, and the cytoplasmic domains may act as a target for negative regulators of αvβ3 activity.

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The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.2994 ◽

1997 ◽

Vol 17 (6) ◽

pp. 2994-3004 ◽

Cited By ~ 31

Author(s):

M Kaouass ◽

M Audette ◽

D Ramotar ◽

S Verma ◽

D De Montigny ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Fusion Gene ◽

Transport Systems ◽

Coding Region ◽

Kinase Gene ◽

Lower Affinity ◽

High Affinity ◽

Polyamine Transport ◽

Exogenous Spermidine

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

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