scholarly journals Resistance to microtubule-stabilising agents following point mutation of human βI-tubulin

2021 ◽  
Author(s):  
◽  
Matthew R. Rowe
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Aspartic Acid ◽  
Mammalian Cells ◽  
Marine Sponges ◽  
Multi Drug Resistance ◽  
Peloruside A ◽  
Characteristic Mode ◽  
Β Tubulin

<p>Marine environments represent a rich source of bioactive secondary metabolites that may be harnessed for use in a therapeutic context. Two novel compounds, peloruside A and laulimalide, isolated from the marine sponges Mycale hentsheli and Cacospongia mycofijiensis, respectively, both demonstrate useful pharmacological properties in mammalian cells. These compounds share major similarities with microtubule-stabilising agents. Like other agents in this class, peloruside A and laulimalide bind to the β-tubulin subunit of microtubules, the primary cytoskeletal element of eukaryotic cells. These compounds enhance polymerisation dynamics between ternary microtubule structures and severely hinder necessary cytoskeletal rearrangements within the cell.  Over the course of a patient’s treatment, cancerous cells may develop multi-drug resistance phenotypes. P-glycoprotein drug efflux pumps play a major role in the development of therapy resistance in many cancers, as the current generation microtubule-stabilising agents are easily removed from diseased cells by upregulated efflux mechanisms. Unlike agents already in clinical application, both peloruside A and laulimalide are poor substrates for removal by these mechanisms, making them and their synthetic derivatives interesting as potential treatments for drug-resistant tumours.  Peloruside A and laulimalide exhibit potent nanomolar anti-mitotic activities in vitro and arrest cell cycle progression in G₂/M phase, leading to cell death – a characteristic mode of action among microtubule-stabilising agents. Unlike all known agents in this class, peloruside A and laulimalide share a secondary, unique binding region in β-tubulin. In the past decade our understanding of this region has developed, revealing a second, unique mechanism for stabilisation of microtubules.  Using mammalian cells to model physiological tubulin, the present study investigates the predicted role of aspartic acid 297 of human βI-tubulin in the binding association of both peloruside A and laulimalide. This particular amino acid is predicted to hydrogen bond with both compounds, contributing to their activity as stabilisers.  It was revealed that the introduction of a point mutation in D297 resulted in a small but highly consistent resistance phenotype to both compounds, but not to microtubule-stabilising agents that bind to the traditional, taxoid site on β-tubulin. It was concluded that aspartic acid 297 is likely to be one of the amino acids directly involved in the binding association of peloruside A and laulimalide to β-tubulin, contributing partial compound stabilisation. The rational synthesis of future analogues may benefit from these findings in the design of molecules with enhanced interactions at this particular amino acid residue.</p>

scholarly journals Resistance to microtubule-stabilising agents following point mutation of human βI-tubulin

2021 ◽  
Author(s):  
◽  
Matthew R. Rowe
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Aspartic Acid ◽  
Mammalian Cells ◽  
Marine Sponges ◽  
Multi Drug Resistance ◽  
Peloruside A ◽  
Characteristic Mode ◽  
Β Tubulin

<p>Marine environments represent a rich source of bioactive secondary metabolites that may be harnessed for use in a therapeutic context. Two novel compounds, peloruside A and laulimalide, isolated from the marine sponges Mycale hentsheli and Cacospongia mycofijiensis, respectively, both demonstrate useful pharmacological properties in mammalian cells. These compounds share major similarities with microtubule-stabilising agents. Like other agents in this class, peloruside A and laulimalide bind to the β-tubulin subunit of microtubules, the primary cytoskeletal element of eukaryotic cells. These compounds enhance polymerisation dynamics between ternary microtubule structures and severely hinder necessary cytoskeletal rearrangements within the cell.  Over the course of a patient’s treatment, cancerous cells may develop multi-drug resistance phenotypes. P-glycoprotein drug efflux pumps play a major role in the development of therapy resistance in many cancers, as the current generation microtubule-stabilising agents are easily removed from diseased cells by upregulated efflux mechanisms. Unlike agents already in clinical application, both peloruside A and laulimalide are poor substrates for removal by these mechanisms, making them and their synthetic derivatives interesting as potential treatments for drug-resistant tumours.  Peloruside A and laulimalide exhibit potent nanomolar anti-mitotic activities in vitro and arrest cell cycle progression in G₂/M phase, leading to cell death – a characteristic mode of action among microtubule-stabilising agents. Unlike all known agents in this class, peloruside A and laulimalide share a secondary, unique binding region in β-tubulin. In the past decade our understanding of this region has developed, revealing a second, unique mechanism for stabilisation of microtubules.  Using mammalian cells to model physiological tubulin, the present study investigates the predicted role of aspartic acid 297 of human βI-tubulin in the binding association of both peloruside A and laulimalide. This particular amino acid is predicted to hydrogen bond with both compounds, contributing to their activity as stabilisers.  It was revealed that the introduction of a point mutation in D297 resulted in a small but highly consistent resistance phenotype to both compounds, but not to microtubule-stabilising agents that bind to the traditional, taxoid site on β-tubulin. It was concluded that aspartic acid 297 is likely to be one of the amino acids directly involved in the binding association of peloruside A and laulimalide to β-tubulin, contributing partial compound stabilisation. The rational synthesis of future analogues may benefit from these findings in the design of molecules with enhanced interactions at this particular amino acid residue.</p>

scholarly journals A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1862-1862
Author(s):  
Gregory J. Cost ◽  
Morayma Temoche-Diaz ◽  
Janet Mei ◽  
Cristina N. Butterfield ◽  
Christopher T. Brown ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Attractive Alternative ◽  
Vitro Assay ◽  
Crispr System ◽  
Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Premature Translation of oskar in Oocytes Lacking the RNA-Binding Protein Bicaudal-C

1998 ◽  
Vol 18 (8) ◽  
pp. 4855-4862 ◽  
Author(s):  
Emma E. Saffman ◽  
Sylvia Styhler ◽  
Katherine Rother ◽  
Weihua Li ◽  
Stéphane Richard ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Point Mutation ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
C Protein ◽  
Kh Domain ◽  
Anterior Posterior

ABSTRACT Bicaudal-C (Bic-C) is required duringDrosophila melanogaster oogenesis for several processes, including anterior-posterior patterning. The gene encodes a protein with five copies of the KH domain, a motif found in a number of RNA-binding proteins. Using antibodies raised against the BIC-C protein, we show that multiple isoforms of the protein exist in ovaries and that the protein, like the RNA, accumulates in the developing oocyte early in oogenesis. BIC-C protein expressed in mammalian cells can bind RNA in vitro, and a point mutation in one of the KH domains that causes a strong Bic-C phenotype weakens this binding. In addition, oskar translation commences prior to posterior localization of oskar RNA inBic-C − oocytes, indicating thatBic-C may regulate oskar translation during oogenesis.

scholarly journals D-aspartic acid is implicated in the control of testosterone production by the vertebrate gonad. Studies on the female green frog, Rana esculenta

1998 ◽  
Vol 157 (2) ◽  
pp. 199-207 ◽  
Author(s):  
MM Di Fiore ◽  
L Assisi ◽  
V Botte ◽  
A D'Aniello
Keyword(s):  
Amino Acid ◽  
Aspartic Acid ◽  
Rana Esculenta ◽  
Ovarian Follicles ◽  
Sexual Cycle ◽  
Green Frog ◽  
Amphibian Species ◽  
Testosterone Production ◽  
Androgen Synthesis

In the present study we report the occurrence of D-aspartic acid (D-Asp) in the ovary of the green frog Rana esculenta and its putative involvement in testosterone production by the gonad. In the ovary, D-Asp concentrations undergo significant variations during the main phases of the sexual cycle. In spawning females (March), its concentration was low (2.5 +/- 1.1 nmol/g ovary) and during the post-reproductive period (June) it increased and reached its peak level (58.0 +/- 10.1 nmol/g) in October. In that month, vitellogenesis occurs in a new set of ovarian follicles and continues until the next spring. The concentrations of D-Asp in the ovary and of testosterone in the ovary and in the plasma were inversely correlated during the reproductive cycle: when endogenous D-Asp was low (March), testosterone was high (36.9 +/- 4.8 ng/g ovary; 23.1 +/- 2.76 ng/ml plasma) and, in contrast, when the D-Asp concentration was high (October), the testosterone concentration was low (0.86 +/- 0.21 ng/g ovary and 5.0 +/- 1.3 ng/ml plasma). In vivo experiments, consisting of injection of D-Asp (2.0 mumol/g body weight) into the dorsal lymphatic sac of adult female frogs, demonstrated that this amino acid accumulates significantly in the ovary. After 3 h, moreover, it caused a decrease in testosterone level in the plasma of about 80%. This inhibition was reversible: within 18 h after the amino acid injection, as the D-Asp concentration in the ovary decreased, the testosterone titre was restored in both ovary and plasma. In vitro experiments, conducted in isolated ovarian follicles, confirmed this phenomenon and identified these gonadal components as the putative D-Asp targets. Other amino acids (L-Asp, D-Glu, L-Glu, D-Ala and L-Ala) used instead of D-Asp were ineffective. These findings indicate that D-Asp is involved in the control of androgen secretion by the ovary in this amphibian species, revealing a more complex system for control of this androgen synthesis than was previously believed to exist.

scholarly journals A feedback transcriptional mechanism controls the level of the arginine/lysine transporter cat-1 during amino acid starvation

2007 ◽  
Vol 402 (1) ◽  
pp. 163-173 ◽  
Author(s):  
Alex B. Lopez ◽  
Chuanping Wang ◽  
Charlie C. Huang ◽  
Ibrahim Yaman ◽  
Yi Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Basic Leucine Zipper

The adaptive response to amino acid limitation in mammalian cells inhibits global protein synthesis and promotes the expression of proteins that protect cells from stress. The arginine/lysine transporter, cat-1, is induced during amino acid starvation by transcriptional and post-transcriptional mechanisms. It is shown in the present study that the transient induction of cat-1 transcription is regulated by the stress response pathway that involves phosphorylation of the translation initiation factor, eIF2 (eukaryotic initiation factor-2). This phosphorylation induces expression of the bZIP (basic leucine zipper protein) transcription factors C/EBP (CCAAT/enhancer-binding protein)-β and ATF (activating transcription factor) 4, which in turn induces ATF3. Transfection experiments in control and mutant cells, and chromatin immunoprecipitations showed that ATF4 activates, whereas ATF3 represses cat-1 transcription, via an AARE (amino acid response element), TGATGAAAC, in the first exon of the cat-1 gene, which functions both in the endogenous and in a heterologous promoter. ATF4 and C/EBPβ activated transcription when expressed in transfected cells and they bound as heterodimers to the AARE in vitro. The induction of transcription by ATF4 was inhibited by ATF3, which also bound to the AARE as a heterodimer with C/EBPβ. These results suggest that the transient increase in cat-1 transcription is due to transcriptional activation caused by ATF4 followed by transcriptional repression by ATF3 via a feedback mechanism.

scholarly journals Two Mutations in β-Tubulin 2 Gene Associated with Thiophanate-Methyl Resistance in Colletotrichum cereale Isolates from Creeping Bentgrass in Mississippi and Alabama

Plant Disease ◽  
2010 ◽  
Vol 94 (2) ◽  
pp. 207-212 ◽  
Author(s):  
Joseph R. Young ◽  
Maria Tomaso-Peterson ◽  
Karla de la Cerda ◽  
Francis P. Wong
Keyword(s):  
Amino Acid ◽  
Creeping Bentgrass ◽  
Colony Growth ◽  
Amino Acid Sequences ◽  
Common Disease ◽  
Thiophanate Methyl ◽  
Colletotrichum Cereale ◽  
In Vitro Bioassays ◽  
Β Tubulin

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass putting greens during the summer in Mississippi and Alabama over the last 15 years. Thiophanate-methyl is a single-site mode-of-action fungicide applied to control C. cereale. In vitro bioassays were performed to evaluate the sensitivity of 103 isolates to thiophanate-methyl concentrations ranging from 0.039 to 10 μg/ml. Eighty-three isolates were collected from creeping bentgrass in Mississippi and Alabama that had been exposed to thiophanate-methyl. An additional 20 isolates were included from nonexposed turfgrasses. Radial colony growth in amended media was relative to nonamended media for all in vitro bioassays. With thiophanate-methyl at 10 μg/ml, relative growth of exposed isolates ranged from 77.5 to 130.7% with a mean of 99.3% compared with nonexposed, baseline isolates that ranged from 0.0 to 48.7% with a mean of 20.4%. A representative sample of thiophanate-methyl-exposed and nonexposed isolates was used to determine the mechanism of resistance by comparing amino acid sequences of the β-tubulin 2 protein. All of the thiophanate-methyl-exposed isolates that were sequenced had a point mutation resulting in substitutions from glutamic acid to alanine at position 198 or from phenylalanine to tyrosine at position 200 of the β-tubulin 2 protein. These amino acid substitutions in C. cereale isolates from Mississippi and Alabama appear to confer resistance to thiophanate-methyl and differ from those reported previously for this pathogen.

scholarly journals Pyridoxine Refractory X-Linked Sideroblastic Anemia Caused by a Point Mutation in the Erythroid 5-Aminolevulinate Synthase Gene

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 822-830 ◽  
Author(s):  
Kazumichi Furuyama ◽  
Hiroyoshi Fujita ◽  
Tadashi Nagai ◽  
Kentaro Yomogida ◽  
Hiroshi Munakata ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Point Mutation ◽  
Bone Marrow Cells ◽  
In Vitro Transcription ◽  
Transient Transfection ◽  
Sideroblastic Anemia ◽  
Aminolevulinate Synthase ◽  
E Protein

Abstract To elucidate how pyridoxine-refractory X-linked sideroblastic anemia (XLSA) develops, we analyzed the erythroid-specific 5-aminolevulinate synthase (ALAS-E) gene of a patient with the anemia. The activity and amount of the enzyme in bone marrow cells of the patient were found to be approximately 5% of the normal control. We identified a point mutation, which introduces an amino acid substitution from Asp 190 to Val. In transient transfection analyses using quail fibroblasts, accumulation of aberrantly processed proteins, the sizes of which were larger than that of mature ALAS-E, was found in mitochondria. The proteins were reproducibly detected in assays combining in vitro transcription/translation of ALAS-E precursor and import of the precursor into isolated mouse mitochondria. These results suggest that the mutation causing pyridoxine-refractory XLSA affects the processing of the ALAS-E precursor, thus provoking instability of the ALAS-E protein.

Inhibition of Hemoglobin Degrading Protease Falcipain-2 as a Mechanism for Anti-Malarial Activity of Triazole-Amino Acid Hybrids

2020 ◽  
Vol 20 (5) ◽  
pp. 377-389 ◽  
Author(s):  
Vigyasa Singh ◽  
Rahul Singh Hada ◽  
Amad Uddin ◽  
Babita Aneja ◽  
Mohammad Abid ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Antimalarial Activity ◽  
Docking Studies ◽  
Dependent Manner ◽  
Trophozoite Stage ◽  
Hemoglobin Degradation

Background: Novel drug development against malaria parasite over old conventional antimalarial drugs is essential due to rapid and indiscriminate use of drugs, which led to the emergence of resistant strains. Methods: In this study, previously reported triazole-amino acid hybrids (13-18) are explored against Plasmodium falciparum as antimalarial agents. Among six compounds, 15 and 18 exhibited antimalarial activity against P. falciparum with insignificant hemolytic activity and cytotoxicity towards HepG2 mammalian cells. In molecular docking studies, both compounds bind into the active site of PfFP-2 and block its accessibility to the substrate that leads to the inhibition of target protein further supported by in vitro analysis. Results: Antimalarial half-maximal inhibitory concentration (IC50) of 15 and 18 compounds were found to be 9.26 μM and 20.62 μM, respectively. Blood stage specific studies showed that compounds, 15 and 18 are effective at late trophozoite stage and block egress pathway of parasites. Decreased level of free monomeric heme was found in a dose dependent manner after the treatment with compounds 15 and 18, which was further evidenced by the reduction in percent of hemoglobin hydrolysis. Compounds 15 and 18 hindered hemoglobin degradation via intra- and extracellular cysteine protease falcipain-2 (PfFP-2) inhibitory activity both in in vitro and in vivo in P. falciparum. Conclusion: We report antimalarial potential of triazole-amino acid hybrids and their role in the inhibition of cysteine protease PfFP-2 as its mechanistic aspect.

Synthesis of aspartic acid derivatives useful for the preparation of misacylated transfer RNAs

2000 ◽  
Vol 78 (6) ◽  
pp. 884-891 ◽  
Author(s):  
Michiel Lodder ◽  
Curtis F Crasto ◽  
Andrei L Laikhter ◽  
Haoyun An ◽  
Tuncer Arslan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Side Chain ◽  
Transfer Rnas ◽  
Rna Ligase ◽  
T4 Rna Ligase ◽  
In Vitro Transcripts ◽  
Derivatives Of

Several derivatives of aspartic acid were protected on Nα as their NVOC derivatives, and on the side chain carboxylates as nitroveratryl esters. Following activation as the cyanomethyl esters, these fully protected aspartate derivatives were converted to the respective pdCpA esters. The protected aspartyl-pdCpA esters were then utilized as substrates for T4 RNA ligase in the presence of in vitro transcripts of tRNA lacking the pCpA dinucleotide normally found at the 3'-end. In this fashion, several misacylated tRNAs were prepared; following photolytic deprotection, these were employed successfully for incorporation into proteins at predetermined positions.Key words: aminoacylated nucleotides, amino acid protection, protein synthesis, tRNA activation.

scholarly journals TAPping into the treasures of tubulin using novel protein production methods

2018 ◽  
Vol 62 (6) ◽  
pp. 781-792
Author(s):  
Nuo Yu ◽  
Niels Galjart
Keyword(s):  
Mammalian Cells ◽  
Gene Families ◽  
Cell Types ◽  
Cellular Functions ◽  
Tubulin Isotypes ◽  
Associated Proteins ◽  
Cytoskeletal Elements ◽  
Different Cell Types ◽  
Β Tubulin

Microtubules are cytoskeletal elements with important cellular functions, whose dynamic behaviour and properties are in part regulated by microtubule-associated proteins (MAPs). The building block of microtubules is tubulin, a heterodimer of α- and β-tubulin subunits. Longitudinal interactions between tubulin dimers facilitate a head-to-tail arrangement of dimers into protofilaments, while lateral interactions allow the formation of a hollow microtubule tube that mostly contains 13 protofilaments. Highly homologous α- and β-tubulin isotypes exist, which are encoded by multi-gene families. In vitro studies on microtubules and MAPs have largely relied on brain-derived tubulin preparations. However, these consist of an unknown mix of tubulin isotypes with undefined post-translational modifications. This has blocked studies on the functions of tubulin isotypes and the effects of tubulin mutations found in human neurological disorders. Fortunately, various methodologies to produce recombinant mammalian tubulins have become available in the last years, allowing researchers to overcome this barrier. In addition, affinity-based purification of tagged tubulins and identification of tubulin-associated proteins (TAPs) by mass spectrometry has revealed the ‘tubulome’ of mammalian cells. Future experiments with recombinant tubulins should allow a detailed description of how tubulin isotype influences basic microtubule behaviour, and how MAPs and TAPs impinge on tubulin isotypes and microtubule-based processes in different cell types.

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