scholarly journals Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3273 ◽  
Author(s):  
David Roura Padrosa ◽  
Jessica Castro ◽  
Alejandro Romero-Casañas ◽  
Marc Ribó ◽  
Maria Vilanova ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Tumor Cells ◽  
Disulfide Bonds ◽  
Site Directed Mutagenesis ◽  
Proteinase K ◽  
Human Sera ◽  
Localization Signal ◽  
Cytotoxic Proteins ◽  
Thermal Stability Of

Ribonucleases are proteins whose use is promising in anticancer therapy. We have previously constructed different human pancreatic ribonuclease variants that are selectively cytotoxic for tumor cells by introducing a nuclear localization signal into their sequence. However, these modifications produced an important decrease in their stability compromising their behavior in vivo. Here, we show that we can significantly increase the thermal stability of these cytotoxic proteins by introducing additional disulfide bonds by site-directed mutagenesis. One of these variants increases its thermal stability by around 17 °C, without affecting its catalytic activity while maintaining the cytotoxic activity against tumor cells. We also show that the most stable variant is significantly more resistant to proteolysis when incubated with proteinase K or with human sera, suggesting that its half-live could be increased in vivo once administered.

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme

2008 ◽  
Vol 389 (9) ◽  
Author(s):  
Hester G. O'Neill ◽  
Pierre Redelinghuys ◽  
Sylva L.U. Schwager ◽  
Edward D. Sturrock
Keyword(s):  
Thermal Stability ◽  
Angiotensin Converting Enzyme ◽  
Physicochemical Properties ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Thermal Stabilization ◽  
Site Directed Mutagenesis ◽  
Converting Enzyme ◽  
Complex Glycans ◽  
Thermal Stability Of

Abstract The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.

Effect of hydration andpH on the thermal stability of proteinase K

1997 ◽  
Vol 50 (1-2) ◽  
pp. 73-80 ◽  
Author(s):  
Wang Bangning ◽  
Han Buxing ◽  
Tan Fu
Keyword(s):  
Thermal Stability ◽  
Proteinase K ◽  
Thermal Stability Of

scholarly journals Disulfide-Mediated Oligomer Formation in Borrelia burgdorferi Outer Surface Protein C, a Critical Virulence Factor and Potential Lyme Disease Vaccine Candidate

2011 ◽  
Vol 18 (6) ◽  
pp. 901-906 ◽  
Author(s):  
Christopher G. Earnhart ◽  
DeLacy V. L. Rhodes ◽  
Richard T. Marconi
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Disulfide Bonds ◽  
Vaccine Candidate ◽  
Site Directed Mutagenesis ◽  
Yield Strain ◽  
Content Type ◽  
Oligomer Formation ◽  
Lyme Disease Vaccine

ABSTRACTBorrelia burgdorferiOspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution amongB. burgdorferisensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization.B. burgdorferiB31ospCwas replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required forin vivofunction, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.

Effects of glycine betaine and glycerophosphocholine on thermal stability of ribonuclease

1998 ◽  
Vol 274 (4) ◽  
pp. F762-F765 ◽  
Author(s):  
Maurice B. Burg ◽  
Eugenia M. Peters
Keyword(s):  
Thermal Stability ◽  
Tissue Culture ◽  
Glycine Betaine ◽  
Metabolic Cost ◽  
Rnase A ◽  
Renal Cells ◽  
And Function ◽  
Thermal Stability Of

Urea in renal medullas is sufficiently high to perturb macromolecules, yet the cells survive and function. The counteracting osmolytes hypothesis holds that methylamines, such as glycine betaine (betaine) and glycerophosphocholine (GPC) in renal medullas, stabilize macromolecules and oppose the effects of urea. Although betaine counteracts effects of urea on macromolecules in vitro and protects renal cells from urea in tissue culture, renal cells accumulate GPC rather than betaine in response to high urea both in vivo and in tissue culture. A proposed explanation is that GPC counteracts urea more effectively than betaine. However, we previously found GPC slightly less effective than betaine in counteracting inhibition of pyruvate kinase activity by urea. To test another macromolecule, we now compare GPC and betaine in counteracting reduction of the thermal stability of RNase A by urea. We find that urea decreases the thermal transition temperature and that betaine and GPC increase it, counteracting urea approximately equally. Therefore, the preference for GPC in response to high urea presumably has some other basis, such as a lower metabolic cost of GPC accumulation.

A comparative analysis of the evolutionary patterning and mechanistic bases of lactate dehydrogenase thermal stability in porcelain crabs, genus Petrolisthes

2001 ◽  
Vol 204 (4) ◽  
pp. 767-776
Author(s):  
J.H. Stillman ◽  
G.N. Somero
Keyword(s):  
Thermal Stability ◽  
Comparative Analysis ◽  
Lactate Dehydrogenase ◽  
Protein Interactions ◽  
Kinetic Properties ◽  
Post Translational Modification ◽  
Extrinsic Factors ◽  
Thermal Stability Of ◽  
Interspecific Diversity

The kinetic properties of orthologous homologs (orthologs) of enzymes are typically correlated with environmental temperatures in species adapted to different thermal regimes, but correlations between adaptation temperature and enzyme thermal stability are less clear. Although the thermal stability of a protein is related chiefly to its primary structure (including post-translational modification), thermal stability can also be altered by extrinsic factors present in the intracellular milieu. Here, we present a comparative analysis of the thermal stability of lactate dehydrogenase (LDH) orthologs from 22 congeneric species of porcelain crab (genera Petrolisthes and Allopetrolisthes) from a broad range of thermal habitats. Interspecific diversity of LDH stability is high: temperatures required for a 50 % loss of activity in 10 min ranged from 65 to 75.5 degrees C, corresponding to half-lives of less than 1 min to more than 3 h at 70 degrees C. Although stability is positively correlated with maximal habitat temperature in some sister taxa, phylogenetic comparative analysis incorporating all 22 species does not indicate that the interspecific diversity of LDH stability represents an adaptive response to current thermal habitats. Examination of the mechanistic bases of LDH stabilization indicates that differences in stability are related both to properties of the LDH molecule itself (intrinsic stability) and to the effects of extrinsic protein(s). Intrinsic differences were shown by the unfolding of structure during heating, as measured by circular dichroism spectroscopy. Stabilizing effects of extrinsic proteins are implied by the results of cellular fractionation experiments that removed low-molecular-mass solutes and proteins from the muscle homogenates. We conclude that the overall structural stability and functional properties of proteins can evolve independently and that in vivo protein-protein interactions can provide another means to regulate protein stability selectively.

NONSPHEROCYTIC CONGENITAL HEMOLYTIC ANEMIA DUE TO A NEW G-6-PD VARIANT: G-6-PD ALHAMBRA

PEDIATRICS ◽  
1970 ◽  
Vol 45 (2) ◽  
pp. 230-235
Author(s):  
Ernest Beutler ◽  
Robert Rosen
Keyword(s):  
Thermal Stability ◽  
Hemolytic Anemia ◽  
Red Cells ◽  
Essentially Normal ◽  
New Variant ◽  
Thermal Stability Of ◽  
Congenital Hemolytic Anemia

An 11-year-old boy was first known to have a hemolytic episode at the age of 8 years, and a diagnosis of nonspherocytic congenital hemolytic anemia due to G-6-PD deficiency was made soon after the hemolytic episode. The red cells of the patient contained a level of G-6-PD activity approximating 10 to 25% of normal. The residual enzyme was partly purified and characterized. It proved to be electrophoretically slow, and was kinetically essentially normal. The thermal stability of the enzyme was reduced and its stability in vivo was markedly diminished. This enzyme represents a new variant which has been named G-6-PD Alhambra.

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