Mechanistic studies of non-canonical amino acid mutagenesis

Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non-canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved incorporation of ncAAs, mechanistic studies of ncAA incorporation by the protein synthesis machinery have tremendous potential for informing and directing such engineering efforts. In this chapter, we describe a set of complementary biochemical and single-molecule fluorescence assays that we have adapted for mechanistic studies of ncAA incorporation. Collectively, these assays provide data that can guide engineering of the protein synthesis machinery to expand the range of ncAAs that can be incorporated into peptides and increase the efficiency with which they can be incorporated, thereby enabling the full potential of ncAA mutagenesis technology to be realized.

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A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

10.1101/330506 ◽

2018 ◽

Author(s):

Martin Y. Ng ◽

Haibo Zhang ◽

Amy Weil ◽

Vijay Singh ◽

Ryan Jamiolkowski ◽

...

Keyword(s):

Protein Synthesis ◽

Single Molecule ◽

Direct Interaction ◽

Vitro Assay ◽

Premature Termination Codons ◽

Protein Synthesis Machinery ◽

Nonsense Suppressor ◽

Nonsense Suppressors ◽

Selection Of

ABSTRACTNonsense suppressors (NonSups) induce “readthrough”, i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly-purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.Table of Contents artwork

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Common therapeutic strategies for prion and Alzheimer’s diseases

Biological Chemistry ◽

10.1515/hsz-2016-0190 ◽

2016 ◽

Vol 397 (11) ◽

pp. 1115-1124 ◽

Cited By ~ 4

Author(s):

Saioa R. Elezgarai ◽

Emiliano Biasini

Keyword(s):

Protein Synthesis ◽

Therapeutic Strategies ◽

Cellular Prion Protein ◽

Protein Isoforms ◽

Therapeutic Interventions ◽

Misfolded Protein ◽

The Past ◽

Protein Synthesis Machinery ◽

Alzheimer’S Diseases ◽

Alzheimer's Diseases

Abstract A number of unexpected pathophysiological connections linking different neurodegenerative diseases have emerged over the past decade. An example is provided by prion and Alzheimer’s diseases. Despite being distinct pathologies, these disorders share several neurotoxic mechanisms, including accumulation of misfolded protein isoforms, stress of the protein synthesis machinery, and activation of a neurotoxic signaling mediated by the cellular prion protein. Here, in addition to reviewing these mechanisms, we will discuss the potential therapeutic interventions for prion and Alzheimer’s diseases that are arising from the comprehension of their common neurodegenerative pathways.

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Mechanistic studies of non-canonical amino acid mutagenesis

Synthetic and Enzymatic Modifications of the Peptide Backbone - Methods in Enzymology ◽

10.1016/bs.mie.2021.05.001 ◽

2021 ◽

pp. 375-428

Author(s):

Rachel C. Fleisher ◽

Nina Michael ◽

Ruben L. Gonzalez

Keyword(s):

Amino Acid ◽

Mechanistic Studies ◽

Canonical Amino Acid ◽

Amino Acid Mutagenesis

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High-Resolution, Low-Voltage SEM of Cell Wall Regeneration of Yeast Shizosaccharomyces Pombe Protoplasts

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103127 ◽

1988 ◽

Vol 46 ◽

pp. 208-211

Author(s):

M. Osumi ◽

N. Yamada ◽

T. Nagatani

Keyword(s):

Electron Microscope ◽

Scanning Electron Microscope ◽

Low Voltage ◽

Cell Wall Regeneration ◽

The Past ◽

The Poor ◽

Probe Size ◽

Beam Damage ◽

Biological Field ◽

Scanning Electron

Even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage, we did not usually operate in the conventional scanning electron microscope at low voltage because of the poor resolution, especially of bioligical specimens. However, the development of the “in-lens” field emission scanning electron microscope (FESEM) has led to marked inprovement in resolution, especially in the range of 1-5 kV, within the past year. The probe size has been cumulated to be 0.7nm in diameter at 30kV and about 3nm at 1kV. We have been trying to develop techniques to use this in-lens FESEM at low voltage (LVSEM) for direct observation of totally uncoated biological specimens and have developed the LVSEM method for the biological field.

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Theodore W. Schultz. The Economics of Being Poor. Oxford: Blackwell. 1993. ix+340 pp.+ Index. Hardbound. £ 40.00.

The Pakistan Development Review ◽

10.30541/v34i1pp.84-85 ◽

1995 ◽

Vol 34 (1) ◽

pp. 84-85

Author(s):

Mir Annice Mahmood

Keyword(s):

Economic Policy ◽

Human Capital ◽

Policy Formulation ◽

The Past ◽

The Poor ◽

Logical Sequence ◽

Standards Of Living ◽

Economic Journals ◽

Material Conditions ◽

Demand For Children

Improving the material conditions of the poor has been the main focus of economic policy formulation for the past fifty years or so. Thus, in this connection, a vast body of literature has been published which deals with such issues as identifying the poor and suggesting remedies to alleviate their lot. The book by Theodore W. Schultz deals specifically with the economics of the poor. The book is primarily a collection of articles the author wrote over a fortyyear period (1950-1990), and these have been published previously in a number of leading economic journals. The articles have been grouped under three headings: "Most People Are Poor"; "Investing in Skills and Knowledge"; and "Effects of Human Capital". The articles basically deal with the concept of human capital. There is a logical sequence to the articles that make up this book; the poor are identified and steps are then suggested to improve their standing. Issues such as women's economic emancipation and the demand for children are highlighted in the collection of articles dealing with these two subjects. By investing in themselves through education, the poor raise their level of skills, and thus their level of wages/salaries, allowing them to enjoy higher standards of living.

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Faculty Opinions recommendation of Probing cellular protein complexes using single-molecule pull-down.

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

10.3410/f.11205956.12310054 ◽

2011 ◽

Author(s):

Michael Ehlers

Keyword(s):

Single Molecule ◽

Protein Complexes ◽

Cellular Protein

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Faculty Opinions recommendation of Probing cellular protein complexes using single-molecule pull-down.

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

10.3410/f.11205956.12190054 ◽

2011 ◽

Author(s):

Manju Hingorani

Keyword(s):

Single Molecule ◽

Protein Complexes ◽

Cellular Protein

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Integrating Bioinformatics Strategies in Cancer Immunotherapy: Current and Future Perspectives

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207323666200427113734 ◽

2020 ◽

Vol 23 (8) ◽

pp. 687-698 ◽

Cited By ~ 1

Author(s):

Houda N. Washah ◽

Elliasu Y. Salifu ◽

Opeyemi Soremekun ◽

Ahmed A. Elrashedy ◽

Geraldene Munsamy ◽

...

Keyword(s):

Cancer Immunotherapy ◽

Therapeutic Potential ◽

Cell Cancer ◽

Full Potential ◽

Future Perspectives ◽

The Past ◽

Bioinformatics Tools ◽

Treatment Paradigm ◽

Immunotherapy Of Cancer ◽

Significant Attention

For the past few decades, the mechanisms of immune responses to cancer have been exploited extensively and significant attention has been given into utilizing the therapeutic potential of the immune system. Cancer immunotherapy has been established as a promising innovative treatment for many forms of cancer. Immunotherapy has gained its prominence through various strategies, including cancer vaccines, monoclonal antibodies (mAbs), adoptive T cell cancer therapy, and immune checkpoint therapy. However, the full potential of cancer immunotherapy is yet to be attained. Recent studies have identified the use of bioinformatics tools as a viable option to help transform the treatment paradigm of several tumors by providing a therapeutically efficient method of cataloging, predicting and selecting immunotherapeutic targets, which are known bottlenecks in the application of immunotherapy. Herein, we gave an insightful overview of the types of immunotherapy techniques used currently, their mechanisms of action, and discussed some bioinformatics tools and databases applied in the immunotherapy of cancer. This review also provides some future perspectives in the use of bioinformatics tools for immunotherapy.

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