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  1. AU="Kyle K. Biggar"
  2. AU="Al-Garawi, Amal"
  3. AU="Freeman, Willard M"
  4. AU="Lussier, A M"
  5. AU="J.Castaneda, "
  6. AU="Izquierdo, Ledys"
  7. AU="Werner, F"
  8. AU="Boddington, Marie E"
  9. AU="N Siddaiah"

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  1. Artikel ; Online: Assessing sequence-based protein–protein interaction predictors for use in therapeutic peptide engineering

    François Charih / Kyle K. Biggar / James R. Green

    Scientific Reports, Vol 12, Iss 1, Pp 1-

    2022  Band 13

    Abstract: Abstract Engineering peptides to achieve a desired therapeutic effect through the inhibition of a specific target activity or protein interaction is a non-trivial task. Few of the existing in silico peptide design algorithms generate target-specific ... ...

    Abstract Abstract Engineering peptides to achieve a desired therapeutic effect through the inhibition of a specific target activity or protein interaction is a non-trivial task. Few of the existing in silico peptide design algorithms generate target-specific peptides. Instead, many methods produce peptides that achieve a desired effect through an unknown mechanism. In contrast with resource-intensive high-throughput experiments, in silico screening is a cost-effective alternative that can prune the space of candidates when engineering target-specific peptides. Using a set of FDA-approved peptides we curated specifically for this task, we assess the applicability of several sequence-based protein–protein interaction predictors as a screening tool within the context of peptide therapeutic engineering. We show that similarity-based protein–protein interaction predictors are more suitable for this purpose than the state-of-the-art deep learning methods publicly available at the time of writing. We also show that this approach is mostly useful when designing new peptides against targets for which naturally-occurring interactors are already known, and that deploying it for de novo peptide engineering tasks may require gathering additional target-specific training data. Taken together, this work offers evidence that supports the use of similarity-based protein–protein interaction predictors for peptide therapeutic engineering, especially peptide analogs.
    Schlagwörter Medicine ; R ; Science ; Q
    Thema/Rubrik (Code) 540
    Sprache Englisch
    Erscheinungsdatum 2022-06-01T00:00:00Z
    Verlag Nature Portfolio
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  2. Artikel ; Online: Evaluation of Jumonji C lysine demethylase substrate preference to guide identification of in vitro substrates

    Matthew Hoekstra / Anand Chopra / William G. Willmore / Kyle K. Biggar

    STAR Protocols, Vol 3, Iss 2, Pp 101271- (2022)

    2022  

    Abstract: Summary: Within the realm of lysine methylation, the discovery of lysine methyltransferase (KMTs) substrates has been burgeoning because of established systematic substrate screening protocols. Here, we describe a protocol enabling the systematic ... ...

    Abstract Summary: Within the realm of lysine methylation, the discovery of lysine methyltransferase (KMTs) substrates has been burgeoning because of established systematic substrate screening protocols. Here, we describe a protocol enabling the systematic identification of JmjC KDM substrate preference and in vitro substrates. Systematically designed peptide libraries containing methylated lysine residues are used to characterize enzyme-substrate preference and identify new candidate substrates in vitro.For complete details on the use and execution of this protocol, please refer to Hoekstra and Biggar (2021).
    Schlagwörter High Throughput Screening ; Molecular Biology ; Protein Biochemistry ; Protein expression and purification ; Systems biology ; Science (General) ; Q1-390
    Sprache Englisch
    Erscheinungsdatum 2022-06-01T00:00:00Z
    Verlag Elsevier
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  3. Artikel ; Online: Single-step purification of intrinsic protein complexes in Saccharomyces cerevisiae using regenerable calmodulin resin

    Urvi Bhojoo / Kyle K. Biggar

    MethodsX, Vol 5, Iss , Pp 613-

    2018  Band 619

    Abstract: Characterization of protein- protein interactions is a vital aspect of molecular biology as multi-protein complexes are the functional units of cellular processes and defining their interactions provides valuable insights into their function based on a “ ... ...

    Abstract Characterization of protein- protein interactions is a vital aspect of molecular biology as multi-protein complexes are the functional units of cellular processes and defining their interactions provides valuable insights into their function based on a “guilt by association” concept. To identify the components in complexes, purification of the latter near homogeneity is required. The tandem affinity purification (i.e., TAP) method, coupled with mass spectrometry have been extensively used to define native protein complexes and transient protein-protein interactions under near physiological conditions (i.e., conditions approximate of the internal milieu) in Saccharomyces cerevisiae. Generally, TAP consists of two-stage protein enrichment using dual affinity tags, a calmodulin-binding peptide and a Staphylococcus aureus protein-A, separated by a tobacco etch virus protease site, which are fused to either the C- or N-terminal of the target protein. TAP-tagging has proved to be a powerful method for studying functional relationship between proteins and generating large-scale protein networks. The method described in this paper provides an inexpensive single-step purification alternative to the traditional two step affinity purification of TAP-tagged proteins using only the calmodulin-binding peptide affinity tag. Moreover, a novel protocol for the regeneration of the calmodulin-agarose resin is outlined and validated. This basic approach allows fast and cost-effective purification of proteins and their interacting partners from Saccharomyces cerevisiae. Keywords: TAP-tag, Matrix regeneration, Saccharomyces cerevisiae, COMPASS complex, Swd3-TAP, Set1
    Schlagwörter Science ; Q
    Thema/Rubrik (Code) 612
    Sprache Englisch
    Erscheinungsdatum 2018-01-01T00:00:00Z
    Verlag Elsevier
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  4. Artikel ; Online: Using Machine Learning and Targeted Mass Spectrometry to Explore the Methyl-Lys Proteome

    Francois Charih / James R. Green / Kyle K. Biggar

    STAR Protocols, Vol 1, Iss 3, Pp 100135- (2020)

    2020  

    Abstract: Summary: Protein lysine methylation mediates a variety of biological processes, and their dysregulation has been established to play pivotal roles in human disease. A number of these sites constitute attractive drug targets. However, systematic ... ...

    Abstract Summary: Protein lysine methylation mediates a variety of biological processes, and their dysregulation has been established to play pivotal roles in human disease. A number of these sites constitute attractive drug targets. However, systematic identification of methylation sites is challenging and resource intensive. Here, we present a protocol combining MethylSight, a machine learning model trained to identify promising lysine methylation sites, and mass spectrometry for subsequent validation. Our approach can reduce the time and investment required to identify novel methylation sites.For complete information on the use and execution of this protocol, please refer to Biggar et al. (2020).
    Schlagwörter Science (General) ; Q1-390
    Sprache Englisch
    Erscheinungsdatum 2020-12-01T00:00:00Z
    Verlag Elsevier
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  5. Artikel ; Online: Multi-schema computational prediction of the comprehensive SARS-CoV-2 vs. human interactome

    Kevin Dick / Anand Chopra / Kyle K. Biggar / James R. Green

    PeerJ, Vol 9, p e

    2021  Band 11117

    Abstract: Background Understanding the disease pathogenesis of the novel coronavirus, denoted SARS-CoV-2, is critical to the development of anti-SARS-CoV-2 therapeutics. The global propagation of the viral disease, denoted COVID-19 (“coronavirus disease 2019”), ... ...

    Abstract Background Understanding the disease pathogenesis of the novel coronavirus, denoted SARS-CoV-2, is critical to the development of anti-SARS-CoV-2 therapeutics. The global propagation of the viral disease, denoted COVID-19 (“coronavirus disease 2019”), has unified the scientific community in searching for possible inhibitory small molecules or polypeptides. A holistic understanding of the SARS-CoV-2 vs. human inter-species interactome promises to identify putative protein-protein interactions (PPI) that may be considered targets for the development of inhibitory therapeutics. Methods We leverage two state-of-the-art, sequence-based PPI predictors (PIPE4 & SPRINT) capable of generating the comprehensive SARS-CoV-2 vs. human interactome, comprising approximately 285,000 pairwise predictions. Three prediction schemas (all, proximal, RP-PPI) are leveraged to obtain our highest-confidence subset of PPIs and human proteins predicted to interact with each of the 14 SARS-CoV-2 proteins considered in this study. Notably, the use of the Reciprocal Perspective (RP) framework demonstrates improved predictive performance in multiple cross-validation experiments. Results The all schema identified 279 high-confidence putative interactions involving 225 human proteins, the proximal schema identified 129 high-confidence putative interactions involving 126 human proteins, and the RP-PPI schema identified 539 high-confidence putative interactions involving 494 human proteins. The intersection of the three sets of predictions comprise the seven highest-confidence PPIs. Notably, the Spike-ACE2 interaction was the highest ranked for both the PIPE4 and SPRINT predictors with the all and proximal schemas, corroborating existing evidence for this PPI. Several other predicted PPIs are biologically relevant within the context of the original SARS-CoV virus. Furthermore, the PIPE-Sites algorithm was used to identify the putative subsequence that might mediate each interaction and thereby inform the design of inhibitory polypeptides ...
    Schlagwörter Protein–protein interaction ; Machine learning ; Comprehensive interactome ; SARS-CoV-2 ; Inter-species interaction prediction ; Medicine ; R ; Biology (General) ; QH301-705.5
    Thema/Rubrik (Code) 570
    Sprache Englisch
    Erscheinungsdatum 2021-04-01T00:00:00Z
    Verlag PeerJ Inc.
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  6. Artikel ; Online: Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol

    Anand Chopra / William G. Willmore / Kyle K. Biggar

    Scientific Reports, Vol 9, Iss 1, Pp 1-

    2019  Band 8

    Abstract: Abstract The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent ... ...

    Abstract Abstract The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.
    Schlagwörter Medicine ; R ; Science ; Q
    Thema/Rubrik (Code) 290
    Sprache Englisch
    Erscheinungsdatum 2019-09-01T00:00:00Z
    Verlag Nature Publishing Group
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  7. Artikel ; Online: Navigating oxygen deprivation

    Kyle K. Biggar / Jing Zhang / Kenneth B. Storey

    PeerJ, Vol 7, p e

    liver transcriptomic responses of the red eared slider turtle to environmental anoxia

    2019  Band 8144

    Abstract: The best facultative anaerobes among vertebrates are members of the genera Trachemys (pond slider turtles) and Chrysemys (painted turtles), and are able to survive without oxygen for up to 12 to 18 weeks at ∼3 °C. In this study, we utilized RNAseq to ... ...

    Abstract The best facultative anaerobes among vertebrates are members of the genera Trachemys (pond slider turtles) and Chrysemys (painted turtles), and are able to survive without oxygen for up to 12 to 18 weeks at ∼3 °C. In this study, we utilized RNAseq to profile the transcriptomic changes that take place in response to 20 hrs of anoxia at 5 °C in the liver of the red eared slide turtle (Trachemys scripta elegans). Sequencing reads were obtained from at least 18,169 different genes and represented a minimum 49x coverage of the C. picta bellii exome. A total of 3,105 genes showed statistically significant changes in gene expression between the two animal groups, of which 971 also exhibited a fold change equal to or greater than 50% of control normoxic values. This study also highlights a number of anoxia-responsive molecular pathways that are may be important to navigating anoxia survival. These pathways were enriched in mRNA found to significantly increase in response to anoxia and included molecular processes such as DNA damage repair and metabolic reprogramming. For example, our results indicate that the anoxic turtle may utilize succinate metabolism to yield a molecule of GTP in addition to the two molecules that results from lactate production, and agrees with other established models of anoxia tolerance. Collectively, our analysis provides a snapshot of the molecular landscape of the anoxic turtle and may provide hints into the how this animal is capable of surviving this extreme environmental stress.
    Schlagwörter Anoxia tolerance ; Hypometabolism ; Metabolic rate depression ; Red eared slider turtle ; DNA damage response ; Medicine ; R ; Biology (General) ; QH301-705.5
    Thema/Rubrik (Code) 612
    Sprache Englisch
    Erscheinungsdatum 2019-11-01T00:00:00Z
    Verlag PeerJ Inc.
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  8. Artikel ; Online: An optimized method using peptide arrays for the identification of in vitro substrates of lysine methyltransferase enzymes

    Elyn M. Rowe / Kyle K. Biggar

    MethodsX, Vol 5, Iss , Pp 118-

    2018  Band 124

    Abstract: While a number of post-translational modifications (PTM), such as phosphorylation and ubiquitination, have been extensively studied, lysine methylation is emerging as an important PTM with implications in a growing number of diverse cellular processes. ... ...

    Abstract While a number of post-translational modifications (PTM), such as phosphorylation and ubiquitination, have been extensively studied, lysine methylation is emerging as an important PTM with implications in a growing number of diverse cellular processes. To date, there are approximately 5000 identified methylation sites on non-histone proteins, and as the methyllysine proteome expands it becomes important to identify the lysine methyltransferase enzymes responsible for each methylation event. The use of peptide SPOT methylation assay has proven to be a useful in the identification and validation of novel substrates for lysine methyltransferase enzymes as it uses a weak beta emitter coupled with fluorography to detect methylation events. The method described in this paper provides improvements to the typical protocol for this assay, as a highly sensitive tritium assay can be developed with less radioactivity than previously described. This protocol provides an inexpensive alternative to weak beta signal enhancer sprays and washes for use in lysine methylation peptide SPOT arrays, and a simple open-source method for array quantification. Protocol name: Highly sensitive in vitro SPOT methylation assay, Keywords: proteomics, non-histone methylation, SMYD3, MAP3K2, VEGFR1
    Schlagwörter Science ; Q
    Thema/Rubrik (Code) 570
    Sprache Englisch
    Erscheinungsdatum 2018-01-01T00:00:00Z
    Verlag Elsevier
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  9. Artikel ; Online: The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans

    Kyle K. Biggar / Kenneth B. Storey

    PeerJ, Vol 6, p e

    2018  Band 4755

    Abstract: In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of ... ...

    Abstract In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.
    Schlagwörter Hypometablism ; Metabolic rate depression ; Cell cycle ; Protein complex ; Medicine ; R ; Biology (General) ; QH301-705.5
    Thema/Rubrik (Code) 570
    Sprache Englisch
    Erscheinungsdatum 2018-05-01T00:00:00Z
    Verlag PeerJ Inc.
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  10. Artikel ; Online: Intriguing Origins of Protein Lysine Methylation

    Natalie Mezey / William C.S. Cho / Kyle K. Biggar

    Genomics, Proteomics & Bioinformatics, Vol 17, Iss 6, Pp 551-

    Influencing Cell Function Through Dynamic Methylation

    2019  Band 557

    Schlagwörter Biology (General) ; QH301-705.5
    Sprache Englisch
    Erscheinungsdatum 2019-12-01T00:00:00Z
    Verlag Elsevier
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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