Functional and Structural Characterization of Nucleic Acid Ligands That Bind to Activated Coagulation Factor XIII

Coagulation factor XIII (FXIII) is a protransglutaminase which plays an important role in clot stabilization and composition by cross-linking the α- and γ-chains of fibrin and increasing the resistance of the clot to mechanical and proteolytic challenges. In this study, we selected six DNA aptamers specific for activated FXIII (FXIIIa) and investigated the functional characterization of FXIIIa after aptamer binding. One of these aptamers, named FA12, efficiently captures FXIIIa even in the presence of zymogenic FXIII subunits. Furthermore, this aptamer inhibits the incorporation of FXIII and α2-antiplasmin (α2AP) into fibrin(ogen) with IC50-values of 38 nM and 17 nM, respectively. In addition to FA12, also another aptamer, FA2, demonstrated significant effects in plasma-based thromboelastometry (rotational thromboelastometry analysis, ROTEM)-analysis where spiking of the aptamers into plasma decreased clot stiffness and elasticity (p < 0.0001). The structure–function correlations determined by combining modeling/docking strategies with quantitative in vitro assays revealed spatial overlap of the FA12 binding site with the binding sites of two FXIII substrates, fibrinogen and α2AP, while FA2 binding sites only overlap those of fibrinogen. Taken together, these features especially render the aptamer FA12 as an interesting candidate molecule for the development of FXIIIa-targeting therapeutic strategies and diagnostic assays.

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The binding sites on fibrin(ogen) for guinea pig liver transglutaminase are similar to those of blood coagulation factor XIII. Characterization of the binding of liver transglutaminase to fibrin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68220-1 ◽

1988 ◽

Vol 263 (28) ◽

pp. 14296-14301 ◽

Cited By ~ 5

Author(s):

K E Achyuthan ◽

A Mary ◽

C S Greenberg

Keyword(s):

Guinea Pig ◽

Blood Coagulation ◽

Binding Sites ◽

Factor Xiii ◽

Coagulation Factor ◽

Pig Liver ◽

Coagulation Factor Xiii ◽

Guinea Pig Liver

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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017 Systemic sclerosis fibroblasts show defective activation by coagulation factor XIII in vitro: implications for impaired wound healing in SSc

Rheumatology ◽

10.1093/rheumatology/kez106.016 ◽

2019 ◽

Vol 58 (Supplement_3) ◽

Author(s):

Anna Leslie ◽

Xu Shiwen ◽

David Abraham ◽

Voon Ong ◽

Christopher Denton

Keyword(s):

Wound Healing ◽

Systemic Sclerosis ◽

Factor Xiii ◽

Coagulation Factor ◽

Impaired Wound Healing ◽

Coagulation Factor Xiii

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Novel deep intronic mutation in the coagulation factor XIII a chain gene leading to unexpected RNA splicing in a patient with factor XIII deficiency

BMC Medical Genetics ◽

10.1186/s12881-019-0944-2 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Jun Deng ◽

Dan Li ◽

Heng Mei ◽

Liang Tang ◽

Hua-fang Wang ◽

...

Keyword(s):

Factor Xiii ◽

Premature Termination ◽

Coagulation Factor ◽

Premature Termination Codon ◽

Termination Codon ◽

Chinese Family ◽

Acceptor Site ◽

Coagulation Factor Xiii ◽

Fxiii Deficiency

Abstract Background Coagulation factor XIII (FXIII) plays an essential role in maintaining hemostasis by crosslinking fibrin. Deficiency in FXIII affects clot stability and increases the risk of severe bleeding. Congenital FXIII deficiency is a rare disease. Recently, we identified a Chinese family with FXIII deficiency and investigated the pathogenesis of congenital FXIII deficiency, contributing non-coding pathogenic variants. Methods We performed common tests, coding sequencing by targeted next-generation sequencing (NGS), whole-genome sequencing and splice-sites prediction algorithms. The pathogenesis was investigated via minigene and nonsense-mediated mRNA decay (NMD) by experiments in vitro. Results The proband is homozygote for a novel deep intronic c.799-12G > A mutation in the F13A1 gene. Through direct sequencing of the minigenes mRNA, we found 10 bases of intron 6 insert in the mRNA of mutant minigenes mRNA. The relative expression of EGFP-F13A1 was higher by suppression of NMD in vitro. Furthermore, we found the proband with enhanced thrombin generation (TG). Conclusion We reported a novel deep intronic c.799-12G > A mutation of F13A1 which produced a new acceptor site and frame shifting during translation introducing a premature termination codon. Our results support the premature termination codon triggered NMD. We need to pay attention to the position of potential alterable splicing sites while counselling and genetic test. The finding of enhanced TG indicated that we should be aware of the risk of thrombosis in patients with FXIII deficiency during replacement therapy.

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Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro

10.1101/2021.11.11.468228 ◽

2021 ◽

Author(s):

Mikhail Ali Hameedi ◽

Erica Teixeira Prates ◽

Michael R Garvin ◽

Irimpan Mathews ◽

B Kirtley Amos ◽

...

Keyword(s):

Hydrogen Bonds ◽

Functional Characterization ◽

In Vitro Assays ◽

Side Chain ◽

Signaling Proteins ◽

Binding Residues ◽

Human Immune ◽

Viral Polyprotein

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like (3CLpro) protease can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.14 Å resolution crystal structure of 3CLpro C145S bound to NEMO226-235 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro-NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for in the pathology of COVID-19.

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