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Expression
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Finally, Gunasekaran et al. (2013) elaborated a novel improved method for the facile production and rapid purification of the functional recombinant Qβ replicase heterotetramer. As followed from this study, the successful expression of the soluble Qβ enzyme depended on the EF-Ts and EF-Tu subunits being coexpressed prior to the expression of the β subunit. Efficient coexpression required two different inducible operons to coordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex was achieved by production of the recombinant S1 protein in a separate host. This approach represented a facile way of producing and purifying large amounts of the soluble and active recombinant Qβ replicase tetramer without the necessity of a His-tag for purification.
TGF-β signaling in testicular development, spermatogenesis, and infertility
Published in Rajender Singh, Molecular Signaling in Spermatogenesis and Male Infertility, 2019
Poonam Mehta, Meghali Joshi, Rajender Singh
All TGF-β ligands, on their binding to the receptor complex, transmit biological information to the cell. The receptor complex is heterotetrameric, consisting of two subunits of each receptor, i.e., type I and type II receptors. These receptors were classified on the basis of structural and functional properties—type I receptor shows higher sequence similarity in the kinase domain as compared to the type II receptor (8). These receptor molecules are characterized by their serine/threonine kinase activity and weaker tyrosine kinase activity (8). In mammals, there are seven type I receptors and five type II receptors. There is another receptor class called a type III receptor in mammals, which is known to regulate TGF-β signaling (8).
Apoptosis and Cell Death
Published in John C Watkinson, Raymond W Clarke, Louise Jayne Clark, Adam J Donne, R James A England, Hisham M Mehanna, Gerald William McGarry, Sean Carrie, Basic Sciences Endocrine Surgery Rhinology, 2018
All caspases are produced as inactive zymogens, and are activated by either autocleavage (in the case of initiator caspases) or other upstream caspases. The procaspase is cleaved in two places – a prodomain is removed and two subunits are generated, a large subunit and a small subunit. These are assembled into the active enzyme which is a heterotetramer with head-to-tail alignment of the subunits. As a result of this, protein electrophoresis detection of the small and/or large subunits is indicative of caspase cleavage. In vivo detection of apoptosis by immunohistochemistry is facilitated by use of antibodies that recognize only the cleaved, active form (Figure 6.1). DNA fragmentation has also been used as a way to detect apoptotic cells. The terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labelling (TUNEL) assay detects both double and single strand breaks by adding labelled dUTP to free 3’-hydroxyl termini of fragmented DNA. The problem with this method is that it picks up DNA fragmentation occurring during necrotic cell death and should therefore be used in conjunction with other methods of apoptosis detection. Alternative in situ ligation (ISL) techniques label only 5’ double-stranded DNA breaks with either blunt ends or a 3’ single base overhang with terminal phosphates, allowing for more specific detection of apoptosis-mediated DNA fragmentation over either necrosis or repairable DNA damage.5
Hb Santa Juana (β 108(G10) Asn > Ser): a low oxygen affinity hemoglobin variant in a family of Bosnian background
Published in Hematology, 2023
N. P. Wildenberg, C. Rossi, A. E. Kulozik, J. B. Kunz
Hemoglobin is a heterotetramer composed of two α- and two β-globin chains, with a total of four heme groups as oxygen binding sites. According to the allosteric two-state-model [2], the heterotetramer can exist in a relaxed R state with high oxygen affinity and a strained T state with low oxygen affinity. Binding of an oxygen molecule to one heme group increases the oxygen affinity of the other subunits. This effect explains the sigmoidal shape of the oxygen binding curve. Hemoglobin variants with decreased oxygen affinity stabilize the T-state and result in increased oxygen delivery to the tissues, while oxygen uptake in the lungs is decreased. The oxygen dissociation curve is right-shifted and characterized by a higher oxygen tension required to reach 50% saturation (p50). However, additional factors contribute to oxygen release into the tissue, such as the concentration of 2,3-bisphosphoglycerate, pH, and CO2 tension. Weak oxygen binding to hemoglobin favors oxygen release into the tissue, suppressing erythropoietin secretion and causing normocytic anemia. Although some patients expressing hemoglobin variants with decreased oxygen affinity present with cyanosis, most are asymptomatic. A low oxygen affinity hemoglobin variant may be suggested by otherwise unexplained reduced pulse oximetry measurements and normocytic anemia.
Bone morphogenetic protein (BMP)9 in cancer development: mechanistic, diagnostic, and therapeutic approaches?
Published in Journal of Drug Targeting, 2023
Ali G. Alkhathami, Mustafa Ryadh Abdullah, Muhjaha Ahmed, Hanan Hassan Ahmed, Sarab W. Alwash, Zahra Muhammed Mahdi, Fahad Alsaikhan, Ayed A. Dera
According to similarities regarding amino acid sequences, BMPs are a family of at least 10 different proteins that belong to the TGF-β superfamily and play crucial roles in skeletal development and maintenance, including BMP1 to BMP15, of which BMP1 is a metalloprotease and is not considered a TGFβ member [7]. BMPs transduce their signals by binding to transmembrane serine-threonine kinase receptors (type I and type II receptors). There are four known BMP type I receptors (ALK1, ALK2, ALK3 (BMPRIA) and ALK6 (BMPRIB)) and three type II receptors (BMPR-II, ActR-IIA and ActR-IIB). The heterotetrameric receptor complexes consist of two molecules of type I and II receptors. Upon BMP binding to receptor complexes, type I receptors are phosphorylated and activated by type II receptors which consequently mediate phosphorylation of downstream signalling molecules, leading to the activation of cytoplasmic SMAD proteins [8]. Three classes of SMAD proteins, including receptor-regulated SMADs (R-SMADs, e.g. SMAD1, 5, and 8), the common mediator SMAD (Co-SMAD, e.g. SMAD4), inhibitory SMADs (I-SMADs, e.g. SMAD6 and 7) are involved in BMPR signalling. BMPs binding to the BMPRs results in SMAD4 translocation into the nucleus, which consequently activates transcription factors such as AP1, RUNX, bZIP, Fox, bHLH, Sp1, IRF7, and Homeodomain. Moreover, BMP may also employ SMAD-independent signalling through activating MAPKs such as ERK, JNK, or p38 MAPK [9].
KCNV2 retinopathy: clinical features, molecular genetics and directions for future therapy
Published in Ophthalmic Genetics, 2020
Thales A. C. De Guimaraes, Michalis Georgiou, Anthony G. Robson, Michel Michaelides
KCNV2 is a 2-exon gene, encoding a 545 amino acid protein, that was first cloned in 2002 (8). It is predominantly expressed in the heart and retina (24). When first described, the protein product was named Kv11.1, rather than Kv8.2, as it is known now; with the nomenclature change being that Kv11.1 was reassigned to a pore-forming subunit of a rapidly activating-delayed rectifier K+ channel, a product of the KCNH2 gene (OMIM #152427). Kv8.2 is a regulatory subunit, which is known to be an electrically “silent” K+ channel subunit when expressed as a homotetramer. Initially, Ottschytsch et al. suggested that it combines with other proteins in heterotetrameric complexes. Indeed, Kv2.1 was found to generate current and promote trafficking of Kv6.3, Kv10.1 and Kv8.2, which supported his hypothesis (8). Through obligatory heteromerization with Kv2.1, Kv8.2 affects cellular excitability potential and alters the K+ current.