scholarly journals A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

1999 ◽  
Vol 341 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Jürgen KIRCHBERGER ◽  
Anke EDELMANN ◽  
Gerhard KOPPERSCHLÄGER ◽  
Jürgen J. HEINISCH
Keyword(s):  
Single Point ◽  
Size Exclusion ◽  
Single Point Mutation ◽  
Β Subunit ◽  
Western Blots ◽  
Α Subunit ◽  
Phosphofructokinase Activity ◽  
Atp Inhibition ◽  
Size Exclusion Hplc

Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded α-subunit) or 868 (for the PFK2-encoded β-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots. Size-exclusion HPLC in conjunction with ELISA detection of the enzyme protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant revealed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the α- or the β-subunit to fructose 2,6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme.

scholarly journals Sulfated non-anticoagulant heparin derivative modifies intracellular hemoglobin, inhibits cell sickling in vitro, and prolongs survival of sickle cell mice under hypoxia

Haematologica ◽  
2021 ◽  
Author(s):  
Osheiza Abdulmalik ◽  
Noureldien H. E. Darwish ◽  
Vandhana Muralidharan-Chari ◽  
Maii Abu Taleb ◽  
Shaker A. Mousa
Keyword(s):  
Sickle Cell ◽  
Inflammatory Responses ◽  
Anticoagulant Activity ◽  
Single Point ◽  
Bleeding Complications ◽  
Single Point Mutation ◽  
Repeated Dosing ◽  
Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases adhesiveness of RBCs to alter the rheological properties of the blood and triggers inflammatory responses, leading to hemolysis and vaso-occlusive crisis sequelae. Unfractionated heparin (UFH) and low-molecular weight heparins (LMWH) have been suggested as treatments to relieve coagulation complications in SCD. However, they are associated with bleeding complications after repeated dosing. An alternative sulfated nonanticoagulant heparin derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectindependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice. S-NACH has been further engineered and structurally enhanced to bind with and modify HbS to directly inhibit sickling, thus employing a multimodal approach. Here, we show that S-NACH can (i) directly engage in Schiff-base reactions with HbS to decrease RBC sickling under both normoxia and hypoxia in vitro, ii) prolong the survival of SCD mice under hypoxia, and (iii) regulate the altered steady state levels of pro- and antiinflammatory cytokines. Thus, our proof of concept in vitro and in vivo preclinical studies demonstrate that the multimodal S-NACH is a highly promising candidate for development into an improved and optimized alternative to LMWHs for the treatment of patients with SCD.

scholarly journals FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ana R. Pereira ◽  
Jen Hsin ◽  
Ewa Król ◽  
Andreia C. Tavares ◽  
Pierre Flores ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Shape ◽  
Single Point ◽  
Spherical Shape ◽  
Single Point Mutation ◽  
Wild Type ◽  
Dead End ◽  
Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

scholarly journals Persistent Equine Arteritis Virus Infection in HeLa Cells

2008 ◽  
Vol 82 (17) ◽  
pp. 8456-8464 ◽  
Author(s):  
Jianqiang Zhang ◽  
Peter J. Timoney ◽  
N. James MacLachlan ◽  
William H. McCollum ◽  
Udeni B. R. Balasuriya
Keyword(s):  
Persistent Infection ◽  
Single Point ◽  
Equine Arteritis Virus ◽  
Structural Proteins ◽  
Single Point Mutation ◽  
Recombinant Viruses ◽  
L Cells ◽  
Passage Number ◽  
Human Cervix

ABSTRACT The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53→Cys and Val55→Ala), GP2 (Leu15→Ser, Trp31→Arg, Val87→Leu, and Ala112→Thr), GP3 (Ser115→Gly and Leu135→Pro), and GP4 (Tyr4→His and Ile109→Phe) proteins or with a single point mutation in the GP5 protein (Pro98→Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

scholarly journals Selective Uncoupling of RGS Action by a Single Point Mutation in the G Protein α-Subunit

1998 ◽  
Vol 273 (10) ◽  
pp. 5780-5784 ◽  
Author(s):  
Paul R. DiBello ◽  
Tiffany Runyan Garrison ◽  
Donald M. Apanovitch ◽  
Ginger Hoffman ◽  
David J. Shuey ◽  
...  
Keyword(s):  
G Protein ◽  
Point Mutation ◽  
Single Point ◽  
Single Point Mutation ◽  
Α Subunit ◽  
G Protein Α Subunit

scholarly journals The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

2000 ◽  
Vol 74 (23) ◽  
pp. 11027-11039 ◽  
Author(s):  
Eran Bacharach ◽  
Jason Gonsky ◽  
Kimona Alin ◽  
Marianna Orlova ◽  
Stephen P. Goff
Keyword(s):  
Leukemia Virus ◽  
Single Point ◽  
Host Protein ◽  
Single Point Mutation ◽  
Gag Protein ◽  
Virion Assembly ◽  
Gag Proteins ◽  
Carboxy Terminal

ABSTRACT A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

scholarly journals Robotic QM/MM-driven maturation of antibody combining sites

2016 ◽  
Vol 2 (10) ◽  
pp. e1501695 ◽  
Author(s):  
Ivan V. Smirnov ◽  
Andrey V. Golovin ◽  
Spyros D. Chatziefthimiou ◽  
Anastasiya V. Stepanova ◽  
Yingjie Peng ◽  
...  
Keyword(s):  
Immune Response ◽  
In Vitro Selection ◽  
Single Point ◽  
Combinatorial Libraries ◽  
Single Point Mutation ◽  
Wild Type ◽  
Selection Of ◽  
Maturation Function

In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

scholarly journals Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

2013 ◽  
Vol 12 (11) ◽  
pp. 1451-1461 ◽  
Author(s):  
Thuy La ◽  
George Desmond Clark-Walker ◽  
Xiaowen Wang ◽  
Stephan Wilkens ◽  
Xin Jie Chen
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Kluyveromyces Lactis ◽  
Β Subunit ◽  
Α Subunit ◽  
Content Type ◽  
Subunit Stoichiometry ◽  
Catalytic Sites ◽  
A Subunit

ABSTRACT F 1 -ATPase is a rotary molecular machine with a subunit stoichiometry of α 3 β 3 γ 1 δ 1 ε 1 . It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α 3 β 3 core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F 1 -ATPase have suggested that the α 3 β 3 core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F 1 -ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ o ) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F 1 complexes retained 21.7 to 44.6% of the native F 1 -ATPase activity. The γ-less F 1 subcomplex was assembled but was structurally and functionally labile in vitro . Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α 3 β 3 subcomplex.

scholarly journals Hypertrophic cardiomyopathy R403Q mutation in rabbit β-myosin reduces contractile function at the molecular and myofibrillar levels

2018 ◽  
Vol 115 (44) ◽  
pp. 11238-11243 ◽  
Author(s):  
Susan Lowey ◽  
Vera Bretton ◽  
Peteranne B. Joel ◽  
Kathleen M. Trybus ◽  
James Gulick ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Contractile Function ◽  
Isometric Force ◽  
Single Point ◽  
Rabbit Model ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Transgenic Rabbits

In 1990, the Seidmans showed that a single point mutation, R403Q, in the human β-myosin heavy chain (MHC) of heart muscle caused a particularly malignant form of familial hypertrophic cardiomyopathy (HCM) [Geisterfer-Lowrance AA, et al. (1990) Cell 62:999–1006.]. Since then, more than 300 mutations in the β-MHC have been reported, and yet there remains a poor understanding of how a single missense mutation in the MYH7 gene can lead to heart disease. Previous studies with a transgenic mouse model showed that the myosin phenotype depended on whether the mutation was in an α- or β-MHC backbone. This led to the generation of a transgenic rabbit model with the R403Q mutation in a β-MHC backbone. We find that the in vitro motility of heterodimeric R403Q myosin is markedly reduced, whereas the actin-activated ATPase activity of R403Q subfragment-1 is about the same as myosin from a nontransgenic littermate. Single myofibrils isolated from the ventricles of R403Q transgenic rabbits and analyzed by atomic force microscopy showed reduced rates of force development and relaxation, and achieved a significantly lower steady-state level of isometric force compared with nontransgenic myofibrils. Myofibrils isolated from the soleus gave similar results. The force–velocity relationship determined for R403Q ventricular myofibrils showed a decrease in the velocity of shortening under load, resulting in a diminished power output. We conclude that independent of whether experiments are performed with isolated molecules or with ordered molecules in the native thick filament of a myofibril, there is a loss-of-function induced by the R403Q mutation in β-cardiac myosin.

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