scholarly journals A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

2021 ◽  
Vol 22 (14) ◽  
pp. 7390
Author(s):  
Nicole Wesch ◽  
Frank Löhr ◽  
Natalia Rogova ◽  
Volker Dötsch ◽  
Vladimir V. Rogov
Keyword(s):  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Biochemical Characterization ◽  
Effective Interaction ◽  
Regulatory Region ◽  
Titration Calorimetry ◽  
Enzymatic Reactions ◽  
Cellular Processes ◽  
Mechanism Of Interaction

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

scholarly journals PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA

2020 ◽  
Author(s):  
Irene Garcia-Maquilon ◽  
Alberto Coego ◽  
Jorge Lozano-Juste ◽  
Maxim Messerer ◽  
Carlos de Ollas ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Molecular Mechanisms ◽  
Date Palm ◽  
Biochemical Characterization ◽  
Soil Water Potential ◽  
Phoenix Dactylifera ◽  
Aba Signaling ◽  
Abiotic Stress Response ◽  
Aba Receptors

Abstract The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant–environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.

scholarly journals Thrombin A-Chain: Activation Remnant or Allosteric Effector?

Thrombosis ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Isis S. R. Carter ◽  
Amanda L. Vanden Hoek ◽  
Edward L. G. Pryzdial ◽  
Ross T. A. MacGillivray
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Current Data ◽  
Enzymatic Reactions ◽  
Allosteric Effector ◽  
Naturally Occurring ◽  
Physiologic Function ◽  
A Chain

Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the other domains. This paper summarizes the current data on the prothrombin catalytic domain A-chain region and the subsequent thrombin A-chain. Attention is given to biochemical characterization of naturally occurring prothrombin A-chain mutations and alanine scanning mutants in this region. While originally considered to be simply an activation remnant with little physiologic function, the thrombin A-chain is now thought to play a role as an allosteric effector in enzymatic reactions and may also be a structural scaffold to stabilize the protease domain.

scholarly journals Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7545
Author(s):  
Jianyou Zhang ◽  
Guangcheng Zhou ◽  
Lifeng Fei ◽  
Lifan Chen ◽  
Lei Sun ◽  
...  
Keyword(s):  
Polyphenol Oxidase ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Enzymatic Reactions ◽  
Inhibitory Effects ◽  
Purification And Characterization ◽  
Effective Prevention ◽  
Ammonium Sulphate Precipitation

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

scholarly journals SapL1: A New Target Lectin for the Development of Antiadhesive Therapy Against Scedosporium apiospermum

2020 ◽  
Author(s):  
Dania Martínez-Alarcón ◽  
Jean-Philippe Bouchara ◽  
Roland J. Pieters ◽  
Annabelle Varrot
Keyword(s):  
Biochemical Characterization ◽  
Present Report ◽  
Binding Mode ◽  
Host Cells ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Scedosporium Apiospermum ◽  
X Ray Crystallography ◽  
Opportunistic Fungal Pathogen

AbstractScedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised patients. This fungus exhibits limited susceptibility to all current antifungals and, due its emerging character, its pathogeny and virulence factors remain largely unknown. Carbohydrate binding proteins such as lectins are involved in host-pathogen interactions and may constitute valuable therapeutic targets to inhibit microbial adhesion to the host cells by using carbohydrate mimics. However, such lectins are still unidentified in S. apiospermum. Here, we present the first report of the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 is homologous to the conidial surface lectin FleA from Aspergillus fumigatus known to be involved in the adhesion to host glycoconjugates present in human lung epithelium. The present report includes a detailed strategy to achieve SapL1 soluble expression in bacteria, its biochemical characterization, an analysis of its specificity and affinity by glycan arrays and isothermal titration calorimetry (ITC), as well as the structural characterization of its binding mode by X–ray crystallography. The information gathered here contribute to the understanding of glycosylated surface recognition by Scedosporium species and is essential for the design and development of antiadhesive glycodrugs targeting SapL1.

scholarly journals NMR Characterization of Conformational Interconversions of Lys48-Linked Ubiquitin Chains

2020 ◽  
Vol 21 (15) ◽  
pp. 5351
Author(s):  
Methanee Hiranyakorn ◽  
Saeko Yanaka ◽  
Tadashi Satoh ◽  
Thunchanok Wilasri ◽  
Benchawan Jityuti ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Hydrophobic Surface ◽  
Enzymatic Reactions ◽  
Multidomain Proteins ◽  
Solvent Exposure ◽  
Hydrophobic Surfaces ◽  
Cellular Processes ◽  
Nmr Characterization

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.

Cellular localization of the microsomal antigen and the thyroid peroxidase antigen

1987 ◽  
Vol 116 (1_Suppl) ◽  
pp. S57-S62 ◽  
Author(s):  
A. Pinchera ◽  
S. Mariotti ◽  
L. Chiovato ◽  
P. Vitti ◽  
G. Lopez ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Human Thyroid ◽  
Thyroid Peroxidase ◽  
Immunofluorescence Analysis ◽  
Thyroid Cells ◽  
Hla Dr ◽  
Microsomal Antigen ◽  
Dependent Mechanism

Abstract. Evidence has been accumulated that human thyroid microsomal/microvillar autoantigen (M) is expressed both in the cytoplasm and on the surface of thyroid follicular cells. The availability of this autoantigen to the immune system, possibly associated with abnormally expressed HLA-DR antigens may be relevant both to the triggering and to maintenance of thyroid autoimmune reactions. Preliminary biochemical characterization of M suggested that it was a glycoprotein with a mol. wt. of about 100–110 kD. recent studies carried out in our laboratories taking advantage of monoclonal antibodies provided evidence that the structure presently referred as M-Ag is represented by thyroid peroxidase (TPO). The identity between TPO and M is further supported by four-layer immunofluorescence analysis showing a complete overlap of the two antigens both in the surface and in the cytoplasm of thyroid cells and by the observation that the expression of M and TPO is similarly modulated by TSH, possibly through a cAMP-dependent mechanism.

scholarly journals Calcium-dependent properties of CIB binding to the integrin αIIb cytoplasmic domain and translocation to the platelet cytoskeleton

1999 ◽  
Vol 342 (3) ◽  
pp. 729-735 ◽  
Author(s):  
David D. SHOCK ◽  
Ulhas P. NAIK ◽  
Julia E. BRITTAIN ◽  
Suresh K. ALAHARI ◽  
John SONDEK ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Cytoplasmic Domain ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Fibrinogen Binding ◽  
Ef Hand ◽  
Domain Peptide ◽  
Triton X

The αIIbβ3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to αIIbβ3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional αIIbβ3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the αIIb cytoplasmic domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed. In addition, isothermal titration calorimetry indicated that CIB binds to an αIIb cytoplasmic-domain peptide in a Ca2+-dependent manner, with moderate affinity (Kd, 700 nM) and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and αIIbβ3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by αIIbβ3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca2+-modulated binding to the αIIb cytoplasmic domain and changes in intracellular distribution.

scholarly journals Myo3A, One of Two Class III Myosin Genes Expressed in Vertebrate Retina, Is Localized to the Calycal Processes of Rod and Cone Photoreceptors and Is Expressed in the Sacculus

2003 ◽  
Vol 14 (3) ◽  
pp. 1058-1073 ◽  
Author(s):  
Andréa C. Dosé ◽  
David W. Hillman ◽  
Cynthia Wong ◽  
Lorraine Sohlberg ◽  
Jennifer Lin-Jones ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Expression Patterns ◽  
Tail Domain ◽  
Class Iii ◽  
Feature Characteristic ◽  
Filament Bundles ◽  
Myosin Motors ◽  
Rod And Cone Photoreceptors

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.

scholarly journals Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae.

1990 ◽  
Vol 171 (5) ◽  
pp. 1535-1546 ◽  
Author(s):  
S A Berish ◽  
T A Mietzner ◽  
L W Mayer ◽  
C A Genco ◽  
B P Holloway ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Neisseria Gonorrhoeae ◽  
Biochemical Characterization ◽  
Iron Acquisition ◽  
Regulatory Region ◽  
Single Copy ◽  
Oligonucleotide Probes ◽  
Gene Encoding

This report describes the cloning and sequencing of the major iron-regulated protein (termed Fbp) of Neisseria gonorrhoeae strain F62. Attempts to identify recombinants expressing the Fbp using specific antibody proved unsuccessful. Therefore, an alternative cloning strategy using oligonucleotide probes derived from NH2-terminal and tryptic fragments of this protein was used to identify short fragments of the gene. Using this methodology, the gene encoding the precursor of Fbp was cloned on three separate overlapping fragments and sequenced, and the amino acid sequence was deduced. These data were unambiguously confirmed by the known NH2-terminal amino acid sequence and were supported by the sequences from tryptic fragments that lie outside of this region. Using oligonucleotide probes, we were unable to obtain clones encoding the potential regulatory region of this protein. Therefore, the technique of inverse polymerase chain reaction was used to amplify a fragment containing an additional 200 bp. This fragment was cloned and sequenced and found to contain a consensus ribosome binding site and potential -10 and -35 sequences. Hybridization analysis of genomic DNA from gonococcal strain F62 indicated that only a single copy of the Fbp gene exists per genome. These results complement the biochemical characterization of the Fbp expressed by gonococci and further suggest that it has a role in iron-acquisition.

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