scholarly journals Age of diabetes onset in the mutant proinsulin syndrome correlates with mutational impairment of protein foldability and stability

2021 ◽  
Author(s):  
Balamurugan Dhayalan ◽  
Yen-Shan Chen ◽  
Yanwu Yang ◽  
Mark Jarosinski ◽  
Deepak Chatterjee ◽  
...  
Keyword(s):  
Molten Globule ◽  
Disease Onset ◽  
Single Chain ◽  
Human Insulin Gene ◽  
Disulfide Pairing ◽  
Single Chain Insulin ◽  
Hormone Analogs ◽  
Α Helix ◽  
Core Packing

Diverse heterozygous mutations in the human insulin gene cause a monogenic diabetes mellitus (DM) syndrome due to toxic misfolding of the variant proinsulin. Whereas mutations that add or remove cysteines (thereby leading to an odd number of thiol groups) generally lead to neonatal-onset DM, non-Cys-related mutations can be associated with a broad range of ages of onset. Here, we compare two mutations at a conserved position in the central B-chain α-helix: one neonatal in DM onset (ValB18→Gly) and the other with onset delayed until adolescence (AlaB18). The substitutions were introduced within a 49-residue single-chain insulin precursor optimized for folding efficiency (Zaykov, A., et al. ACS Chem. Biol. 9, 683-91 (2014)). Although mutations are each unfavorable, GlyB18 (a) more markedly perturbs DesDi folding efficiency in vitro than does AlaB18 and (b) more severely induces endoplasmic reticulum (ER) stress in cell-based studies of the respective proinsulin variants. In corresponding two-chain hormone analogs, GlyB18 more markedly perturbs structure, function and thermodynamic stability than does AlaB18. Indeed, the GlyB18-insulin analog forms a molten globule with attenuated α-helix content whereas the AlaA18 analog retains a nativelike cooperative structure with reduced free energy of unfolding (ΔΔGu 1.2(±0.2) kcal/mole relative to ValB18 parent). We propose that mutations at B18 variably impede nascent pairing of CysB19 and CysA20 to an extent correlated with perturbed core packing once native disulfide pairing is achieved. Differences in age of disease onset (neonatal or adolescent) reflect relative biophysical perturbations (severe or mild) of an obligatory on-pathway protein folding intermediate.

In Vitro Folding/Unfolding of Insulin/Single-Chain Insulin

2006 ◽  
Vol 13 (5) ◽  
pp. 423-429 ◽  
Author(s):  
Zhi-Song Qiao ◽  
Zhan-Yun Guo ◽  
You-Min Feng
Keyword(s):  
Single Chain ◽  
Single Chain Insulin

scholarly journals Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite

2004 ◽  
Vol 186 (6) ◽  
pp. 1861-1868 ◽  
Author(s):  
Lingyun Song ◽  
Jonathan Caguiat ◽  
Zhongrui Li ◽  
Jacob Shokes ◽  
Robert A. Scott ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Metal Binding ◽  
Coiled Coil ◽  
Mercury Resistance ◽  
Binding Motif ◽  
Single Chain ◽  
Metal Binding Domain ◽  
Α Helix

ABSTRACT The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more 203Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step.

Fibrin-specificity of a Plasminogen Activator Affects the Efficiency of Fibrinolysis and Responsiveness to Ultrasound: Comparison of Nine Plasminogen Activators In Vitro

1999 ◽  
Vol 81 (04) ◽  
pp. 605-612 ◽  
Author(s):  
Dmitry V. Sakharov ◽  
Marrie Barrett-Bergshoeff ◽  
Rob T. Hekkenberg ◽  
Dingeman C. Rijken
Keyword(s):  
Thrombolytic Therapy ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Bolus Administration ◽  
High Frequency Ultrasound ◽  
Single Chain ◽  
Response Curves ◽  
Maximal Speed ◽  
Frequency Ultrasound

SummaryIn a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed.Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot.In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Absence of Synergism Between Tissue-Type Plasminogen Activator (t-PA), Single-Chain UrokinaseType Plasminogen Activator (scu-PA) and Urokinase on Clot Lysis in a Plasma Milieu In Vitro

1986 ◽  
Vol 56 (01) ◽  
pp. 035-039 ◽  
Author(s):  
D Collen ◽  
F De Cock ◽  
E Demarsin ◽  
H R Lijnen ◽  
D C Stump
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Dose Response ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryA potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scuPA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in titrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and α2-antiplasmin.t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant α2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal.

Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro

2019 ◽  
Vol 19 (5) ◽  
pp. 610-619 ◽  
Author(s):  
Xue-Qing Zhang ◽  
Lu-Ting Yu ◽  
Pei Du ◽  
Tian-Qi Yin ◽  
Zhi-Yuan Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Cell Death ◽  
Cancer Cell ◽  
Gastric Cancer Cell ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ags Cells ◽  
Thiazolyl Blue Tetrazolium Bromide

Background:Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein.Methods:This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay.Results:The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed.Conclusion:The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

scholarly journals Development of a bispecific immune engager using a recombinant malaria protein

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Mie A. Nordmaj ◽  
Morgan E. Roberts ◽  
Emilie S. Sachse ◽  
Robert Dagil ◽  
Anne Poder Andersen ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Immune Evasion ◽  
Blood Cells ◽  
Xenograft Model ◽  
Cancer Targeting ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Conjugation System ◽  
Immune Mediated

AbstractAs an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

scholarly journals Astrocytes Are More Vulnerable than Neurons to Silicon Dioxide Nanoparticle Toxicity in Vitro

Toxics ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 51
Author(s):  
Jorge Humberto Limón-Pacheco ◽  
Natalie Jiménez-Barrios ◽  
Alejandro Déciga-Alcaraz ◽  
Adriana Martínez-Cuazitl ◽  
Mónica Maribel Mata-Miranda ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Silicon Dioxide ◽  
Culture Media ◽  
Brain Cell ◽  
Attenuated Total Reflection ◽  
Random Coil ◽  
Silicon Dioxide Nanoparticles ◽  
Neurotoxic Effects ◽  
Α Helix

Some studies have shown that silicon dioxide nanoparticles (SiO2-NPs) can reach different regions of the brain and cause toxicity; however, the consequences of SiO2-NPs exposure on the diverse brain cell lineages is limited. We aimed to investigate the neurotoxic effects of SiO2-NP (0–100 µg/mL) on rat astrocyte-rich cultures or neuron-rich cultures using scanning electron microscopy, Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), FTIR microspectroscopy mapping (IQ mapping), and cell viability tests. SiO2-NPs were amorphous particles and aggregated in saline and culture media. Both astrocytes and neurons treated with SiO2-NPs showed alterations in cell morphology and changes in the IR spectral regions corresponding to nucleic acids, proteins, and lipids. The analysis by the second derivative revealed a significant decrease in the signal of the amide I (α-helix, parallel β-strand, and random coil) at the concentration of 10 µg/mL in astrocytes but not in neurons. IQ mapping confirmed changes in nucleic acids, proteins, and lipids in astrocytes; cell death was higher in astrocytes than in neurons (10–100 µg/mL). We conclude that astrocytes were more vulnerable than neurons to SiO2-NPs toxicity. Therefore, the evaluation of human exposure to SiO2-NPs and possible neurotoxic effects must be followed up.

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