A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.

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De novo structural mutation rates and gamete-of-origin biases revealed through genome sequencing of 2,396 families

10.1101/2020.10.06.329011 ◽

2020 ◽

Author(s):

Jonathan R. Belyeu ◽

Harrison Brand ◽

Harold Wang ◽

Xuefang Zhao ◽

Brent S. Pedersen ◽

...

Keyword(s):

De Novo ◽

Genome Structure ◽

Point Mutations ◽

Large Family ◽

Germline Mutations ◽

Autism Spectrum ◽

Parental Age ◽

Single Nucleotide Variants ◽

Multiple Testing Correction ◽

Structural Mutations

AbstractEach human genome includes de novo mutations that arose during gametogenesis. While these germline mutations represent a fundamental source of new genetic diversity, they can also create deleterious alleles that impact fitness. The germline mutation rate for single nucleotide variants and factors that significantly influence this rate, such as parental age, are now well established. However, far less is known about the frequency, distribution, and features that impact de novo structural mutations. We report a large, family-based study of germline mutations, excluding aneuploidy, that affect genome structure among 572 genomes from 33 families in a multigenerational CEPH-Utah cohort and 2,363 cases of non-familial autism spectrum disorder (ASD), 1,938 unaffected siblings, and both parents (9,599 genomes in total). We find that de novo structural mutations detected by alignment-based, short-read WGS occurred at an overall rate of at least 0.160 events per genome in unaffected individuals and was significantly higher (0.206 per genome) in ASD cases. In both probands and unaffected samples, nearly 73% of de novo structural mutations arose in paternal gametes, and predict most de novo structural mutations to be caused by mutational mechanisms that do not require sequence homology. After multiple testing correction we did not observe a statistically significant correlation between parental age and the rate of de novo structural variation in offspring. These results highlight that a spectrum of mutational mechanisms contribute to germline structural mutations, and that these mechanisms likely have markedly different rates and selective pressures than those leading to point mutations.

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Faculty Opinions recommendation of Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1013669.191606 ◽

2003 ◽

Author(s):

Anne Bowcock

Keyword(s):

De Novo ◽

Point Mutations ◽

Lamin A ◽

Progeria Syndrome ◽

Hutchinson Gilford Progeria Syndrome

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Targeting HER2 Alterations in Non–Small-Cell Lung Cancer: A Comprehensive Review

JCO Precision Oncology ◽

10.1200/po.19.00333 ◽

2020 ◽

pp. 411-425 ◽

Cited By ~ 6

Author(s):

Jing Zhao ◽

Yang Xia

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Kinase Inhibitors ◽

Antitumor Agents ◽

De Novo ◽

Point Mutations ◽

Small Cell ◽

Small Cell Lung ◽

Exon 20

PURPOSE HER2 is a critical gene that drives various solid tumors in addition to those of breast cancer. For example, HER2 plays a role in non–small-cell lung cancer (NSCLC). Overexpression, amplification, and point mutations in HER2 have been described in patients with NSCLC; however, the potential roles of these alterations remain unclear. METHODS We summarize the evidence regarding the distinct impacts of different HER2 aberrations on antitumor agents. Also, we update the therapeutic efficacy of HER2-targeted agents, including anti-HER2 antibodies, antibody-drug conjugates, and small-molecule tyrosine kinase inhibitors, tested in HER2-aberrant NSCLC. RESULTS Although these drugs are not yet standard treatments, certain patients may benefit from these therapies. In this review, we aim to provide an improved understanding of HER2 aberrations in NSCLC, including NSCLC biology and the impacts of each aberration on prognosis and standard treatment. We also highlight the potential of novel anti-HER2 therapies approved by regulatory bodies and those in clinical development. CONCLUSION Compared with HER2 amplification or overexpression, HER2 mutations, especially HER2 exon 20 mutations, are emerging as the most clear targetable driver for HER2-directed therapies in lung cancer. De novo and inducible HER2 pathway activation need to be differentially managed. Further investigations with new strategies are needed.

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Structure of SPH (self-incompatibility protein homologue) proteins: a widespread family of small, highly stable, secreted proteins

Biochemical Journal ◽

10.1042/bcj20180828 ◽

2019 ◽

Vol 476 (5) ◽

pp. 809-826

Author(s):

Karthik V. Rajasekar ◽

Shuangxi Ji ◽

Rachel J. Coulthard ◽

Jon P. Ride ◽

Gillian L. Reynolds ◽

...

Keyword(s):

Fusion Protein ◽

Analytical Ultracentrifugation ◽

De Novo ◽

Learning Algorithm ◽

Sequence Similarity ◽

Homology Modelling ◽

3D Structure ◽

Large Family ◽

Secreted Proteins ◽

Self Incompatibility

Abstract SPH (self-incompatibility protein homologue) proteins are a large family of small, disulfide-bonded, secreted proteins, initially found in the self-incompatibility response in the field poppy (Papaver rhoeas), but now known to be widely distributed in plants, many containing multiple members of this protein family. Using the Origami strain of Escherichia coli, we expressed one member of this family, SPH15 from Arabidopsis thaliana, as a folded thioredoxin fusion protein and purified it from the cytosol. The fusion protein was cleaved and characterised by analytical ultracentrifugation, circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. This showed that SPH15 is monomeric and temperature stable, with a β-sandwich structure. The four strands in each sheet have the same topology as the unrelated proteins: human transthyretin, bacterial TssJ and pneumolysin, with no discernible sequence similarity. The NMR-derived structure was compared with a de novo model, made using a new deep learning algorithm based on co-evolution/correlated mutations, DeepCDPred, validating the method. The DeepCDPred de novo method and homology modelling to SPH15 were then both used to derive models of the 3D structure of the three known PrsS proteins from P. rhoeas, which have only 15–18% sequence homology to SPH15. The DeepCDPred method gave models with lower discreet optimised protein energy scores than the homology models. Three loops at one end of the poppy structures are postulated to interact with their respective pollen receptors to instigate programmed cell death in pollen tubes.

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Biocatalysis by Transglutaminases: A Review of Biotechnological Applications

Micromachines ◽

10.3390/mi9110562 ◽

2018 ◽

Vol 9 (11) ◽

pp. 562 ◽

Cited By ~ 9

Author(s):

Maria Savoca ◽

Elisa Tonoli ◽

Adeola Atobatele ◽

Elisabetta Verderio

Keyword(s):

Nutritive Value ◽

Large Family ◽

Catalytic Triad ◽

Matrix Proteins ◽

Primary Amines ◽

Biotechnological Applications ◽

Post Translational Modification ◽

The Past ◽

Lysine Residues ◽

Isopeptide Bonds

The biocatalytic activity of transglutaminases (TGs) leads to the synthesis of new covalent isopeptide bonds (crosslinks) between peptide-bound glutamine and lysine residues, but also the transamidation of primary amines to glutamine residues, which ultimately can result into protein polymerisation. Operating with a cysteine/histidine/aspartic acid (Cys/His/Asp) catalytic triad, TGs induce the post-translational modification of proteins at both physiological and pathological conditions (e.g., accumulation of matrices in tissue fibrosis). Because of the disparate biotechnological applications, this large family of protein-remodelling enzymes have stimulated an escalation of interest. In the past 50 years, both mammalian and microbial TGs polymerising activity has been exploited in the food industry for the improvement of aliments’ quality, texture, and nutritive value, other than to enhance the food appearance and increased marketability. At the same time, the ability of TGs to crosslink extracellular matrix proteins, like collagen, as well as synthetic biopolymers, has led to multiple applications in biomedicine, such as the production of biocompatible scaffolds and hydrogels for tissue engineering and drug delivery, or DNA-protein bio-conjugation and antibody functionalisation. Here, we summarise the most recent advances in the field, focusing on the utilisation of TGs-mediated protein multimerisation in biotechnological and bioengineering applications.

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Genotyping structural variants in pangenome graphs using the vg toolkit

10.1101/654566 ◽

2019 ◽

Cited By ~ 7

Author(s):

Glenn Hickey ◽

David Heller ◽

Jean Monlong ◽

Jonas A. Sibbesen ◽

Jouni Sirén ◽

...

Keyword(s):

De Novo ◽

State Of The Art ◽

Effective Means ◽

Point Mutations ◽

Structural Variants ◽

Short Read ◽

Yeast Strains ◽

Sequencing Studies ◽

Long Read

AbstractStructural variants (SVs) remain challenging to represent and study relative to point mutations despite their demonstrated importance. We show that variation graphs, as implemented in the vg toolkit, provide an effective means for leveraging SV catalogs for short-read SV genotyping experiments. We benchmarked vg against state-of-the-art SV genotypers using three sequence-resolved SV catalogs generated by recent long-read sequencing studies. In addition, we use assemblies from 12 yeast strains to show that graphs constructed directly from aligned de novo assemblies improve genotyping compared to graphs built from intermediate SV catalogs in the VCF format.

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De Novo Mutational Signature Discovery in Tumor Genomes using SparseSignatures

10.1101/384834 ◽

2018 ◽

Cited By ~ 5

Author(s):

Avantika Lal ◽

Keli Liu ◽

Robert Tibshirani ◽

Arend Sidow ◽

Daniele Ramazzotti

Keyword(s):

Cross Validation ◽

De Novo ◽

State Of The Art ◽

Point Mutations ◽

Simulated Data ◽

Large Datasets ◽

Genome Sequences ◽

Mutational Signatures ◽

Mutational Signature ◽

Current State

AbstractCancer is the result of mutagenic processes that can be inferred from tumor genomes by analyzing rate spectra of point mutations, or “mutational signatures”. Here we present SparseSignatures, a novel framework to extract signatures from somatic point mutation data. Our approach incorporates DNA replication error as a background, employs regularization to reduce noise in non-background signatures, uses cross-validation to identify the number of signatures, and is scalable to large datasets. We show that SparseSignatures outperforms current state-of-the-art methods on simulated data using standard metrics. We then apply SparseSignatures to whole genome sequences of 147 tumors from pancreatic cancer, discovering 8 signatures in addition to the background.

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Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Viruses ◽

10.3390/v14010146 ◽

2022 ◽

Vol 14 (1) ◽

pp. 146

Author(s):

Angelo Pavesi ◽

Fabio Romerio

Keyword(s):

De Novo ◽

Point Mutations ◽

Selective Advantage ◽

Genomic Region ◽

Fine Tuning ◽

Sequence Evolution ◽

Viral Genomes ◽

Stop Codons ◽

Hiv 1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

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Unprecedented η3-Coordination and Functionalization of the 2-Phosphaethynthiolate Anion at Lanthanum(III)

10.26434/chemrxiv.13567400 ◽

2021 ◽

Author(s):

Fabian A. Watt ◽

Lukas Burkhardt ◽

Roland Schoch ◽

Stefan Mitzinger ◽

Matthias Bauer ◽

...

Keyword(s):

Reaction Mechanism ◽

Dft Calculations ◽

Coordination Mode ◽

Quantum Mechanical ◽

Structural Comparison ◽

Anion Complex ◽

Steric Factors ◽

Group 15 ◽

Detailed Reaction Mechanism ◽

The Ideal

We present the unprecedented η3-coordination of the 2-phosphaethynthiolate anion in the complex (PN)2La(SCP) (2) [PN = N-(2-(diisopropylphosphanyl)-4-methylphenyl)-2,4,6-trimethylanilide)]. Structural comparison with dinuclear thiocyanate bridged (PN)2La(μ-1,3-SCN)2La(PN)2 (3) and azide bridged (PN)2La(μ-1,3-N3)2La(PN)2 (4) complexes indicates that the [SCP]– coordination mode is mainly governed by electronic, rather than steric factors. Quantum mechanical investigations reveal large contributions of the antibonding π-orbital of the [SCP]– ligand to the LUMO of complex 2, rendering it the ideal precursor for the first functionalization of the [SCP]– anion. Complex 2 was therefore reacted with CAACs which induced a selective rearrangement of the [SCP]– ligand to form the first CAAC stabilized group 15 – group 16 fulminate-type complexes (PN)2La{SPC(RCAAC)} (5a,b) (R = Ad, Me). A detailed reaction mechanism for the SCP to SPC isomerization is proposed based on DFT calculations.

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Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome

Microbiology ◽

10.1099/mic.0.25834-0 ◽

2003 ◽

Vol 149 (8) ◽

pp. 2243-2250 ◽

Cited By ~ 18

Author(s):

Shin-ichi Miyata ◽

Kenro Oshima ◽

Shigeyuki Kakizawa ◽

Hisashi Nishigawa ◽

Hee-Young Jung ◽

...

Keyword(s):

Catalytic Activity ◽

Genomic Library ◽

De Novo ◽

Bacterial Species ◽

Point Mutations ◽

Kinase Gene ◽

Thymidylate Kinase ◽

Pcr Products ◽

Multiple Copies ◽

Salvage Pathways

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the onion yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects. Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene. Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations. The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity. Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity. This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome.

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