scholarly journals Tuning the ion selectivity of two-pore channels

2017 ◽  
Vol 114 (5) ◽  
pp. 1009-1014 ◽  
Author(s):  
Jiangtao Guo ◽  
Weizhong Zeng ◽  
Youxing Jiang
Keyword(s):  
Sequence Similarity ◽  
Ion Selectivity ◽  
Structural Basis ◽  
Monovalent Cations ◽  
Nonselective Cation Channel ◽  
High Sequence Similarity ◽  
Selective Channel ◽  
Tm Domain ◽  
Key Residues

Organellar two-pore channels (TPCs) contain two copies of aShaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region, yet exhibit drastically different ion selectivity. Plant TPC1 functions as a nonselective cation channel on the vacuole membrane, whereas mammalian TPC channels have been shown to be endo/lysosomal Na+-selective or Ca2+-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 fromArabidopsis thaliana(AtTPC1) and compared its selectivity with the selectivity of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is selective for Ca2+over Na+, but nonselective among monovalent cations (Li+, Na+, and K+). Our results also confirm that HsTPC2 is a Na+-selective channel activated by phosphatidylinositol 3,5-bisphosphate. Guided by our recent structure of AtTPC1, we converted AtTPC1 to a Na+-selective channel by mimicking the selectivity filter of HsTPC2 and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Na+-selective AtTPC1 mutant elucidates the structural basis for Na+selectivity in mammalian TPCs.

scholarly journals Inheritance of seedlessness and the molecular characterization of the INO gene in Annonaceae

2023 ◽  
Vol 83 ◽  
Author(s):  
B. R. R. M. Nassau ◽  
P. S. C. Mascarenhas ◽  
A. G. Guimarães ◽  
F. M. Feitosa ◽  
H. M. Ferreira ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Type A ◽  
Molecular Techniques ◽  
Wild Type ◽  
Annona Squamosa ◽  
High Sequence Similarity ◽  
Breeding Programs ◽  
Complete Dominance ◽  
Young Leaves

Abstract The inheritance of the seedless fruit characteristic of Annona squamosa has not yet been explained. Molecular techniques may aid breeding programs, mainly in the assisted selection of the target gene. The INO gene may be related to seed development in these fruits. The objective of the present paper was to investigate the inheritance of seedlessness in the 'Brazilian seedless' sugar apple and INO gene conservation in Annona squamosa and Annona cherimola x Annona squamosa genotypes by assessing their homology with the INO database genes. The F1 generation was obtained by crossing the mutant 'Brazilian seedless' (male genitor) (P1) with the wild-type A. squamosa with seeds (M1 and M2, female genitors). The INO gene was studied in mutant and wild-type A. squamosa (P1, M1, M2 and M3) and in the Gefner atemoya (A. cherimola x A. squamosa) (M4) cultivar. The DNA was extracted from young leaves, and four sets of specific primers flanking the INO gene were amplified. The seedless characteristic was identified as stenospermatic in the fruits of parental P1, suggesting monogenic inheritance with complete dominance. High sequence similarity of the INO gene amplifications in the sugar apple accessions (M1, M2, M3) and the atemoya cultivar Gefner (M4) reinforces the hypothesis of their conservation.

scholarly journals Isolation and Characterization of Insecticidal Toxins from the Venom of the North African Scorpion, Buthacus leptochelys

Toxins ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 236 ◽  
Author(s):  
Yusuke Yoshimoto ◽  
Masahiro Miyashita ◽  
Mohammed Abdel-Wahab ◽  
Moustafa Sarhan ◽  
Yoshiaki Nakagawa ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Scorpion Venom ◽  
Spectrometric Analysis ◽  
North African ◽  
High Sequence Similarity ◽  
The North ◽  
Isolation And Characterization ◽  
Ms Analysis ◽  
Insect Toxicity

Various bioactive peptides have been identified in scorpion venom, but there are many scorpion species whose venom has not been investigated. In this study, we characterized venom components of the North African scorpion, Buthacus leptochelys, by mass spectrometric analysis and evaluated their insect toxicity. This is the first report of chemical and biological characterization of the B. leptochelys venom. LC/MS analysis detected at least 148 components in the venom. We isolated four peptides that show insect toxicity (Bl-1, Bl-2, Bl-3, and Bl-4) through bioassay-guided HPLC fractionation. These toxins were found to be similar to scorpion α- and β-toxins based on their N-terminal sequences. Among them, the complete primary structure of Bl-1 was determined by combination of Edman degradation and MS/MS analysis. Bl-1 is composed of 67 amino acid residues and crosslinked with four disulfide bonds. Since Bl-1 shares high sequence similarity with α-like toxins, it is likely that it acts on Na+ channels of both insects and mammals.

scholarly journals Structural insights into the ion selectivity of the MgtE channel for Mg2+ over Ca2+

2021 ◽  
Author(s):  
Xinyu Teng ◽  
Danqi Sheng ◽  
Jin Wang ◽  
Ye Yu ◽  
Motoyuki Hattori
Keyword(s):  
Metal Binding ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Structural Basis ◽  
Recognition Mechanism ◽  
High Resolution Structure ◽  
Moderate Resolution ◽  
Tm Domain ◽  
Functional Analyses ◽  
Computational Analyses

MgtE is a Mg2+-selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and play an important role in the maintenance of cellular Mg2+ homeostasis. Previous functional analyses showed that MgtE transports divalent cations with high selectivity for Mg2+ over Ca2+. Whereas the high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg2+ ions revealed a Mg2+ recognition mechanism of MgtE, the previous Ca2+-bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 angstrom resolution), which was insufficient to visualize the water molecules coordinated to Ca2+ ions. Thus, the structural basis of the ion selectivity of MgtE for Mg2+ over Ca2+ has remained unclear. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg2+ ~500-fold more strongly than Ca2+. We then determined the crystal structure of the MgtE TM domain in complex with Ca2+ ions at a higher resolution (2.5 angstrom resolution), allowing us to reveal hexahydrated Ca2+, which is similarly observed in the previously determined Mg2+-bound structure but with extended metal-oxygen bond lengths. Our structural, biochemical, and computational analyses provide mechanistic insights into the ion selectivity of MgtE for Mg2+ over Ca2+.

scholarly journals Isolation and structural characterization of insulin and glucagon from the holocephalan species Callorhynchus milii (elephantfish)

1989 ◽  
Vol 263 (1) ◽  
pp. 261-266 ◽  
Author(s):  
B C Berks ◽  
C J Marshall ◽  
A Carne ◽  
S M Galloway ◽  
J F Cutfield
Keyword(s):  
Gas Phase ◽  
Receptor Binding ◽  
Structural Characterization ◽  
Sequence Similarity ◽  
Cartilaginous Fish ◽  
Proteolytic Processing ◽  
Reverse Phase ◽  
High Sequence Similarity ◽  
Processing Mechanisms

Both insulin and glucagon from the pancreas of the holocephalan cartilaginous fish Callorhynchus milii (elephantfish) have been isolated and purified. Two reverse-phase h.p.l.c. steps enabled recovery of sufficient material for gas-phase sequencing of the intact chains as well as peptide digestion products. The elephantfish insulin sequence shows 14 differences from pig insulin, including two unusual substitutions, Val-A14 and Gln-B30, though none of these is thought likely to influence receptor binding significantly. The insulin B-chain contains 31 residues, one more than mammalian insulins, but markedly less than that of the closely related ratfish with which it otherwise exhibits high sequence similarity. Elephantfish and pig glucagons differ at only four positions, but there are six changes from the ratfish glucagon-36 (normal glucagon contains 29 residues) sequence. It is apparent that different prohormone proteolytic processing mechanisms operate in the two holocephalan species.

scholarly journals Characterization of a new family of cyclin-dependent kinase activators

2005 ◽  
Vol 386 (2) ◽  
pp. 349-355 ◽  
Author(s):  
Ana DINARINA ◽  
Laurent H. PEREZ ◽  
Amparo DAVILA ◽  
Markus SCHWAB ◽  
Tim HUNT ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Sequence Similarity ◽  
Cdk Inhibitor ◽  
Cell Cycle Regulators ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinases ◽  
The Core ◽  
New Family ◽  
Key Residues

Progression through the cell cycle is regulated by CDKs (cyclin-dependent kinases), which associate with activating partners, named cyclins, to efficiently phosphorylate substrates. We previously reported the identification of RINGO, a Xenopus protein that can activate CDK1 and CDK2 despite lack of sequence similarity to cyclins, which plays a role in the regulation of the meiotic cell cycle in oocytes. In the present study we report the characterization of four mammalian RINGO proteins, which are 53–68% identical with Xenopus RINGO in a central core of about 75 residues. We show that all RINGO family members can bind to and activate CDK1 and CDK2, albeit with different efficiencies, but they do not bind to CDK4 or CDK6. The core RINGO sequences are critical for CDK activation. We also identified key residues in CDK2 that are required for RINGO binding. All RINGO proteins can also bind the CDK inhibitor p27Kip1, but with an inverse efficiency of their ability to bind to CDK1. Our results identify a new family of mammalian proteins that can activate CDKs and therefore potentially function as cell cycle regulators. The ability of RINGO proteins to activate CDK1 and CDK2 suggest also cyclin-independent roles for these kinases.

scholarly journals Voltage-Dependent Anion Selective Channel 3: Unraveling Structural and Functional Features of the Least Known Porin Isoform

2022 ◽  
Vol 12 ◽  
Author(s):  
Simona Reina ◽  
Vanessa Checchetto
Keyword(s):  
Current Knowledge ◽  
Sequence Similarity ◽  
Physiological Role ◽  
Research Field ◽  
Mass Spectrometry Data ◽  
Intermembrane Space ◽  
High Sequence Similarity ◽  
Voltage Dependent ◽  
Selective Channel ◽  
Functional Features

Voltage-dependent anion-selective channels (VDAC) are pore-forming proteins located in the outer mitochondrial membrane. Three isoforms are encoded by separate genes in mammals (VDAC1-3). These proteins play a crucial role in the cell, forming the primary interface between mitochondrial and cellular metabolisms. Research on the role of VDACs in the cell is a rapidly growing field, but the function of VDAC3 remains elusive. The high-sequence similarity between isoforms suggests a similar pore-forming structure. Electrophysiological analyzes revealed that VDAC3 works as a channel; however, its gating and regulation remain debated. A comparison between VDAC3 and VDAC1-2 underlines the presence of a higher number of cysteines in both isoforms 2 and 3. Recent mass spectrometry data demonstrated that the redox state of VDAC3 cysteines is evolutionarily conserved. Accordingly, these residues were always detected as totally reduced or partially oxidized, thus susceptible to disulfide exchange. The deletion of selected cysteines significantly influences the function of the channel. Some cysteine mutants of VDAC3 exhibited distinct kinetic behavior, conductance values and voltage dependence, suggesting that channel activity can be modulated by cysteine reduction/oxidation. These properties point to VDAC3 as a possible marker of redox signaling in the mitochondrial intermembrane space. Here, we summarize our current knowledge about VDAC3 predicted structure, physiological role and regulation, and possible future directions in this research field.

scholarly journals The Isolation of Pyrroloformamide Congeners and Characterization of Its Biosynthetic Gene Cluster from Streptomyces sp. CB02980 Revealed a Unified Mechanism for Dithiolopyrrolone Biosynthesis

2019 ◽  
Author(s):  
Wenqing Zhou ◽  
Haoyu Liang ◽  
Xiangjing Qin ◽  
Danfeng Cao ◽  
Xiangcheng Zhu ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Sequence Similarity ◽  
Biosynthetic Gene Cluster ◽  
Gene Replacement ◽  
Biosynthetic Gene ◽  
High Sequence Similarity ◽  
Bicyclic Structure ◽  
Microbial Natural Products ◽  
Drug Leads

Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. In the current study, two new dithiolopyrrolones, pyrroloformamide C (<b>3</b>) and pyrroloformamide D (<b>4</b>), were isolated from <i>Streptomyces </i>sp. CB02980, together with the known pyrroloformamides <b>1 </b>and <b>2</b>. The biosynthetic gene cluster for pyrroloformamides was identified from <i>S</i>. sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of pyfE, which encodes a non-ribosomal peptide synthetase, abolished the production of <b>1</b>－<b>4</b>. Overexpression of <i>pyfN</i>, a type II thioesterase gene, increased the production of <b>1</b> and <b>2</b>. The structure elucidation and biosynthetic characterization of pyrroloformamides <b>1</b> - <b>4</b> may inspire future efforts to discover new dithiolopyrrolones, which are promising drug leads for the treatment of infectious diseases or cancer.

scholarly journals Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold

2000 ◽  
Vol 348 (3) ◽  
pp. 649-656 ◽  
Author(s):  
Allan M. TORRES ◽  
Greg M. DE PLATER ◽  
Magnus DOVERSKOG ◽  
Liesl C. BIRINYI-STRACHAN ◽  
Graham M. NICHOLSON ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
High Sequence Similarity ◽  
Natural Function ◽  
Global Fold ◽  
Key Residues ◽  
Β Sheet

The venom of the male Australian duck-billed platypus contains a family of four polypeptides of appox. 5 kDa, which are referred to as defensin-like peptides (DLPs). They are unique in that their amino acid sequences have no significant similarities to those of any known peptides; however, the tertiary structure of one of them, DLP-1, has recently been shown to be similar to β-defensin-12 and to the sodium neurotoxin peptide ShI (Stichodactyla helianthus neurotoxin I). Although DLPs are the major peptides in the platypus venom, little is known about their biological roles. In this study, we determined the three-dimensional structure of DLP-2 by NMR spectroscopy, with the aim of gaining insights into the natural function of the DLPs in platypus venom. The DLP-2 structure was found to incorporate a short helix that spans residues 9-12, and an antiparallel β-sheet defined by residues 15-18 and 37-40. The overall fold and cysteine-pairing pattern of DLP-2 were found to be similar to those of DLP-1, and hence β-defensin-12; however, the sequence similarities between the three molecules are relatively small. The distinct structural fold of the DLP-1, DLP-2, and β-defensin-12 is based upon several key residues that include six cysteines. DLP-3 and DLP-4 are also likely to be folded similarly since they have high sequence similarity with DLP-2. The DLPs, and β-defensin-12 may thus be grouped together into a class of polypeptide molecules which have a common or very similar global fold. The fact that the DLPs did not display antimicrobial, myotoxic, or cell-growth-promoting activities implies that the nature of the side chains in this group of peptides is likely to play an important role in defining the biological function(s).

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