scholarly journals Lysosomal protein thermal stability does not correlate with cellular half-life: global observations and a case study of tripeptidyl-peptidase 1

2020 ◽  
Vol 477 (3) ◽  
pp. 727-745 ◽  
Author(s):  
Aaron M. Collier ◽  
Yuliya Nemtsova ◽  
Narendra Kuber ◽  
Whitney Banach-Petrosky ◽  
Anurag Modak ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Engineering ◽  
Half Life ◽  
Neuronal Ceroid Lipofuscinosis ◽  
Turnover Rates ◽  
Lysosomal Storage ◽  
Enzyme Replacement ◽  
Tripeptidyl Peptidase ◽  
The Stability

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.

scholarly journals Protein thermal stability does not correlate with cellular half-life: Global observations and a case study of tripeptidyl-peptidase 1

2019 ◽  
Author(s):  
Aaron M. Collier ◽  
Yuliya Nemtsova ◽  
Narendra Kuber ◽  
Whitney Banach-Petrosky ◽  
Anurag Modak ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Engineering ◽  
Half Life ◽  
Neuronal Ceroid Lipofuscinosis ◽  
Turnover Rates ◽  
Lysosomal Storage ◽  
Enzyme Replacement ◽  
Tripeptidyl Peptidase ◽  
The Stability

AbstractLate-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL-patient derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.

scholarly journals In vitro pharmacological profiling of selected small molecules compound using recombinant human iduronate-2-sulphatase

2021 ◽  
Vol 5 (2) ◽  
pp. 26-30
Author(s):  
Affandi Omar ◽  
Dyg Pertiwi Abg Kamaludin ◽  
Salina Abdul Rahman ◽  
Rosnani Mohamed ◽  
Fatimah Diana Amin Nordin ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Small Molecules ◽  
Heparan Sulphate ◽  
Stability Study ◽  
Lysosomal Storage ◽  
Pharmacological Chaperone ◽  
Enzyme Replacement ◽  
Mps Ii ◽  
System Manifestation

Background: Mucopolysaccharidoses type II (MPS II) is an X-linked lysosomal storage disease (LSD). It is due to mutation in IDS gene encoding iduronate-2-sulphatase (IDS) involved in the catabolism of dermatan sulphate and heparan sulphate. Currently, the treatments for MPS II patients are enzyme replacement therapy (ERT) and bone marrow transplantation (BMT). However, ERT is not effectively reducing the central nervous system manifestation and finding the suitable donor maybe quite challenging in BMT. Over the past decades, pharmacological chaperone has been an alternative approach for management of MPS II patient. Here, we described the in vitro profiling of small molecules in group of chondroitin/dermatan (CD) sulphate disaccharide, heparin oligosaccharides, unsaturated heparin disaccharides and 6-O-desulphated heparin oligosaccharide, using recombinant human iduronate-2-sulphatase (rhIDS). Twenty-one small molecule compounds with several concentrations were each screened by inhibition and thermal stability assays. Results: Our study revealed that condroitin dermatan trisulphate (CD3S), heparin tetrasaccharide (H4Sac), heparin octasaccharide (H8Sac) and heparin octadecasaccharide (H18Sac) showed high inhibition constant, Ki and low inhibition concentration, IC50 in comparison to others. In the thermal stability study, only rhIDS incubated with CD3S was found to preserve enzyme activity (20%) after incubated at 67oC. Conclusion: Overall, our experiments discovered that CD3S was able to bind, inhibit and chaperone rhIDS. These features suggest a potential pharmacological chaperone for MPS II.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

scholarly journals Fabry Disease Therapy: State-of-the-Art and Current Challenges

2020 ◽  
Vol 22 (1) ◽  
pp. 206
Author(s):  
Olga Azevedo ◽  
Miguel Fernandes Gago ◽  
Gabriel Miltenberger-Miltenyi ◽  
Nuno Sousa ◽  
Damião Cunha
Keyword(s):  
Fabry Disease ◽  
State Of The Art ◽  
Large Body ◽  
Real Life ◽  
Clinical Manifestations ◽  
Substrate Reduction Therapy ◽  
Lysosomal Storage ◽  
New Therapies ◽  
Enzyme Replacement

Fabry disease (FD) is a lysosomal storage disorder caused by mutations of the GLA gene that lead to a deficiency of the enzymatic activity of α-galactosidase A. Available therapies for FD include enzyme replacement therapy (ERT) (agalsidase alfa and agalsidase beta) and the chaperone migalastat. Despite the large body of literature published about ERT over the years, many issues remain unresolved, such as the optimal dose, the best timing to start therapy, and the clinical impact of anti-drug antibodies. Migalastat was recently approved for FD patients with amenable GLA mutations; however, recent studies have raised concerns that “in vitro” amenability may not always reflect “in vivo” amenability, and some findings on real-life studies have contrasted with the results of the pivotal clinical trials. Moreover, both FD specific therapies present limitations, and the attempt to correct the enzymatic deficiency, either by enzyme exogenous administration or enzyme stabilization with a chaperone, has not shown to be able to fully revert FD pathology and clinical manifestations. Therefore, several new therapies are under research, including new forms of ERT, substrate reduction therapy, mRNA therapy, and gene therapy. In this review, we provide an overview of the state-of-the-art on the currently approved and emerging new therapies for adult patients with FD.

scholarly journals Immobilization of Mutant Phosphotriesterase on Fuller’s Earth Enhanced the Stability of the Enzyme

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 983
Author(s):  
Wahhida Latip ◽  
Victor Feizal Knight ◽  
Ong Keat Khim ◽  
Noor Azilah Mohd Kasim ◽  
Wan Md Zin Wan Yunus ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Metal Ions ◽  
Half Life ◽  
Industrial Applications ◽  
Free Enzyme ◽  
High Tolerance ◽  
Tween 40 ◽  
Fuller’S Earth ◽  
The Stability ◽  
Biochemical Features

Immobilization is a method for making an enzyme more robust in the environment, especially in terms of its stability and reusability. A mutant phosphotriesterase (YT PTE) isolated from Pseudomonas dimunita has been reported to have high proficiency in hydrolyzing the Sp and Rp-enantiomers of organophosphate chromophoric analogs and therefore has great potential as a decontamination agent and biosensor. This work aims to investigate the feasibility of using Fuller’s earth (FE) as a YT PTE immobilization support and characterize its biochemical features after immobilization. The immobilized YT PTE was found to show improvement in thermal stability with a half-life of 24 h compared to that of the free enzyme, which was only 8 h. The stability of the immobilized YT PTE allowed storage for up to 4 months and reuse for up to 6 times. The immobilized YT PTE showed high tolerance against all tested metal ions, Tween 40 and 80 surfactants and inorganic solvents. These findings showed that the immobilized YT PTE became more robust for use especially with regards to its stability and reusability. These features would enhance the future applicability of this enzyme as a decontamination agent and its use in other suitable industrial applications.

scholarly journals Brain-targeted enzyme-loaded nanoparticles: A breach through the blood-brain barrier for enzyme replacement therapy in Krabbe disease

2019 ◽  
Vol 5 (11) ◽  
pp. eaax7462 ◽  
Author(s):  
Ambra Del Grosso ◽  
Marianna Galliani ◽  
Lucia Angella ◽  
Melissa Santi ◽  
Ilaria Tonazzini ◽  
...  
Keyword(s):  
Nervous System ◽  
Enzymatic Activity ◽  
Krabbe Disease ◽  
Brain Targeting ◽  
Cell Trafficking ◽  
Lysosomal Storage ◽  
Cns Involvement ◽  
Enzyme Replacement ◽  
Activity Measurements

Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement.

scholarly journals Proteomic Analysis in Morquio A Cells Treated with Immobilized Enzymatic Replacement Therapy on Nanostructured Lipid Systems

2019 ◽  
Vol 20 (18) ◽  
pp. 4610 ◽  
Author(s):  
J. Víctor Álvarez ◽  
Susana B. Bravo ◽  
María García-Vence ◽  
María J. De Castro ◽  
Asteria Luzardo ◽  
...  
Keyword(s):  
Replacement Therapy ◽  
Delivery System ◽  
Skeletal Dysplasia ◽  
Cellular Protein ◽  
Lysosomal Storage ◽  
Enzyme Replacement ◽  
Morquio A ◽  
Mps Iva ◽  
Mucopolysaccharidosis Type Iva

Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disease due to mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Systemic skeletal dysplasia and the related clinical features of MPS IVA are due to disruption of cartilage and its extracellular matrix, leading to an imbalance of growth. Enzyme replacement therapy (ERT) with recombinant human GALNS, alpha elosulfase, provides a systemic treatment. However, this therapy has a limited impact on skeletal dysplasia because the infused enzyme cannot penetrate cartilage and bone. Therefore, an alternative therapeutic approach to reach the cartilage is an unmet challenge. We have developed a new drug delivery system based on a nanostructure lipid carrier with the capacity to immobilize enzymes used for ERT and to target the lysosomes. This study aimed to assess the effect of the encapsulated enzyme in this new delivery system, using in vitro proteomic technology. We found a greater internalization of the enzyme carried by nanoparticles inside the cells and an improvement of cellular protein routes previously impaired by the disease, compared with conventional ERT. This is the first qualitative and quantitative proteomic assay that demonstrates the advantages of a new delivery system to improve the MPS IVA ERT.

Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component

1989 ◽  
Vol 9 (5) ◽  
pp. 1996-2006
Author(s):  
G Brewer ◽  
J Ross
Keyword(s):  
Nucleic Acid ◽  
Micrococcal Nuclease ◽  
Turnover Rates ◽  
Acid Component ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
Order Of Magnitude ◽  
The Stability ◽  
Nucleic Acid Component

The turnover rates of some mRNAs vary by an order of magnitude or more when cells change their growth pattern or differentiate. To identify regulatory factors that might be responsible for this variability, we investigated how cytosolic fractions affect mRNA decay in an in vitro system. A 130,000 X g supernatant (S130) from the cytosol of exponentially growing erythroleukemia cells contains a destabilizer that accelerates the decay of polysome-bound c-myc mRNA by eightfold or more compared with reactions lacking S130. The destabilizer is deficient in or absent from the S130 of cycloheximide-treated cells, indicating that it is labile or is repressed when translation is blocked. It is not a generic RNase, because it does not affect the turnover of delta-globin, gamma-globin, or histone mRNA and does not destabilize a major portion of polysomal polyadenylated mRNA. The destabilizer accelerates the turnover of the c-myc mRNA 3' region, as well as subsequent 3'-to-5' degradation of the mRNA body. It is inactivated in vitro by mild heating and by micrococcal nuclease, suggesting that it contains a nucleic acid component. c-myb mRNA is also destabilized in S130-supplemented in vitro reactions. These results imply that the stability of some mRNAs is regulated by cytosolic factors that are not associated with polysomes.

scholarly journals Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

2013 ◽  
Vol 80 (4) ◽  
pp. 1291-1298 ◽  
Author(s):  
Yi Cao ◽  
Jie Li ◽  
Na Jiang ◽  
Xiuzhu Dong
Keyword(s):  
Half Life ◽  
Content Type ◽  
Methanosarcina Mazei ◽  
Reverse Transcription Quantitative Pcr ◽  
Aceticlastic Methanogenesis ◽  
The Stability ◽  
Zoige Wetland ◽  
Mrna Half Life

ABSTRACTMethylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats,Methanosarcina mazeizm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances ofmtaA1,mtaB1, andmtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, whileackAandptamRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. Thein vivohalf-lives ofmtaA1andmtaC1B1mRNAs were similar in 30°C and 15°C cultures. However, thepta-ackAmRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified formtaA1andmtaC1B1mRNAs, while only a 27-nt 5′ UTR was present in thepta-ackAtranscript. Removal of the 5′ UTRs significantly reduced thein vitrohalf-lives ofmtaA1andmtaC1B1mRNAs. Remarkably, fusion of themtaA1ormtaC1B15′ UTRs topta-ackAmRNA increased itsin vitrohalf-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptiveM. mazeizm-15.

scholarly journals Stabilization of the mRNA for the uncoupling protein thermogenin by transcriptional/translational blockade and by noradrenaline in brown adipocytes differentiated in culture: a degradation factor induced by cessation of stimulation?

1994 ◽  
Vol 302 (1) ◽  
pp. 81-86 ◽  
Author(s):  
C Picó ◽  
D Herron ◽  
A Palou ◽  
A Jacobsson ◽  
B Cannon ◽  
...  
Keyword(s):  
Half Life ◽  
Uncoupling Protein ◽  
Mrna Level ◽  
Direct Interaction ◽  
Brown Adipocytes ◽  
Degradation Factor ◽  
The Stability ◽  
High Level

The stability of the mRNA coding for the uncoupling protein thermogenin was investigated in mouse brown-fat cells differentiated in culture. After 7 days in culture, the cells were stimulated for 24 h with noradrenaline, and a high level of thermogenin mRNA was then observed. If noradrenaline treatment was continued, the mRNA level remained high, but, upon withdrawal of noradrenaline, the level decreased rapidly, with a half-life of only 2.7 h. The presence of transcriptional (actinomycin) or translational (cycloheximide) inhibitors prolonged the apparent half-life by about 50%. The presence of noradrenaline during transcriptional blockade led to a further stabilization of thermogenin mRNA. It was concluded that an induced (or short-lived) gene product is important for thermogenin mRNA degradation. Direct interaction of noradrenaline with the cultured brown adipocytes could apparently not mimic the paradoxical destabilization of thermogenin mRNA in vivo, previously observed in the cold-exposed mouse [Jacobsson, Cannon and Nedergaard (1987) FEBS Lett. 244, 353-356], indicating significant differences between the systems in vitro and in vivo.

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