The WH1 and EVH1 Domains of WASP and Ena/VASP Family Members Bind Distinct Sequence Motifs

AbstractMacrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

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Epitope Mapping of Antibodies against Prostate-specific Antigen with Use of Peptide Libraries

Clinical Chemistry ◽

10.1093/clinchem/48.12.2208 ◽

2002 ◽

Vol 48 (12) ◽

pp. 2208-2216 ◽

Cited By ~ 18

Author(s):

Jari Leinonen ◽

Ping Wu ◽

Ulf-Håkan Stenman

Keyword(s):

Prostate Specific Antigen ◽

Cyclic Peptides ◽

Epitope Mapping ◽

Specific Antigen ◽

Peptide Libraries ◽

Sequence Motifs ◽

Large Panel ◽

Distinct Sequence ◽

Binding Peptides ◽

Important Marker

Abstract Background: Prostate-specific antigen (PSA) is the most important marker for prostate cancer, but PSA concentrations determined by various assays can differ significantly because of differences in specificity of the antibodies used. To identify epitopes recognized by various monoclonal antibodies (MAbs) to PSA, we have isolated peptides that react with the paratopes of these. Methods: Six anti-PSA MAbs representing three major epitope groups were screened with five cyclic phage display peptide libraries. After selection, the peptide sequences were determined by sequencing of the relevant part of viral DNA. Binding of the phage peptides to the MAbs was monitored by immunoassay. Results: For each MAb, several paratope-binding peptides with distinct sequence motifs were identified, but only ∼10% showed similarity with the PSA sequence. Some of these correctly predicted the location of the epitopes. By sequential panning of the library with two closely related MAbs, we identified peptides reacting equally with both MAbs. When analyzed against a large panel of PSA MAbs, the peptides generally showed restricted specificity toward the MAb used for selection, but some peptides bound to several related MAbs. Conclusions: Most of the cyclic peptides selected with PSA MAbs are specific for the MAb used for selection and do not resemble any sequence on the antigen. Peptides reactive with two MAbs recognizing the same epitope can be obtained by sequential panning. This method can be used to predict the location of some epitopes, but additional methods are needed to confirm the result.

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Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members

Nucleic Acids Research ◽

10.1093/nar/gkaa161 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4081-4099 ◽

Cited By ~ 3

Author(s):

Alex I Finnegan ◽

Somang Kim ◽

Hu Jin ◽

Michael Gapinske ◽

Wendy S Woods ◽

...

Keyword(s):

Dna Methylation ◽

Time Course ◽

Cytosine Methylation ◽

Family Members ◽

Dynamic Network ◽

Dna Methyltransferases ◽

Genome Wide ◽

Distinct Sequence ◽

Adenosyl Methionine ◽

Methylation Patterns

Abstract Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT’s activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.

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Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages

Journal of Virology ◽

10.1128/jvi.77.10.5557-5570.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5557-5570 ◽

Cited By ~ 124

Author(s):

Carine Ménard ◽

Markus Wagner ◽

Zsolt Ruzsics ◽

Karina Holak ◽

Wolfram Brune ◽

...

Keyword(s):

Gene Family ◽

Family Members ◽

Cell Types ◽

Murine Cytomegalovirus ◽

Sequence Motifs ◽

Gene Deletions ◽

Conserved Sequence ◽

Growth Properties ◽

Growth Phenotype

ABSTRACT The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.

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Sequence signatures of two public antibody clonotypes that bind SARS-CoV-2 receptor binding domain

Nature Communications ◽

10.1038/s41467-021-24123-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Timothy J. C. Tan ◽

Meng Yuan ◽

Kaylee Kuzelka ◽

Gilberto C. Padron ◽

Jacob R. Beal ◽

...

Keyword(s):

Antibody Response ◽

Receptor Binding ◽

Somatic Hypermutation ◽

Binding Domain ◽

Light Chains ◽

Receptor Binding Domain ◽

Sequence Motifs ◽

Major Function ◽

Distinct Sequence ◽

Complementarity Determining Region

AbstractSince the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short complementarity-determining region (CDR) H3. Germline-encoded sequence motifs in heavy chain CDRs H1 and H2 have a major function, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, is not clear. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that seem to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. These results advance understanding of the antibody response to SARS-CoV-2.

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Distinct evolutionary trajectories in asexual populations through an interplay of their size, resource availability and mutation rates

10.1101/2020.08.27.269829 ◽

2020 ◽

Author(s):

Bhaskar Kumawat ◽

Ramray Bhat

Keyword(s):

Mutation Rate ◽

Single Cells ◽

Maximum Size ◽

Mutation Rates ◽

Genome Replication ◽

Sequence Motifs ◽

Complex Picture ◽

Distinct Sequence ◽

Resource Levels ◽

Asexual Populations

AbstractAsexually reproducing populations of single cells evolve through mutation, natural selection, and genetic drift to enhance their reproductive fitness. The environment provides the contexts that allow and regulate their fitness dynamics. In this work, we used Avida - a digital evolution framework - to uncover the effect of mutation rates, maximum size of the population, and the relative abundance of resources, on evolutionary outcomes in asexually reproducing populations of digital organisms. We observed that over extended simulations, the population evolved predominantly to one of several discrete fitness classes, each with distinct sequence motifs and/or phenotypes. For a low mutation rate, the organisms acquired either of four fitness values through an enhancement in the rate of genomic replication. Evolution at a relatively higher mutation rate presented a more complex picture. While the highest fitness values at a high mutation rate were achieved through enhanced genome replication rates, a suboptimal one was achieved through organisms sharing information relevant to metabolic tasks with each other. The information sharing capacity was vital to fitness acquisition and frequency of the genotype associated with it increased with greater resource levels and maximum population size. In addition, populations optimizing their fitness through such means exhibited a greater degree of genotypic heterogeneity and metabolic activity than those that improved replication rates. Our results reveal a minimal set of conditions for the emergence of interdependence within evolving populations with significant implications for biological systems in appropriate environmental contexts.

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Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members

10.1101/2020.01.26.919936 ◽

2020 ◽

Author(s):

Alex I. Finnegan ◽

Somang Kim ◽

Hu Jin ◽

Michael Gapinske ◽

Wendy S. Woods ◽

...

Keyword(s):

Dna Methylation ◽

Time Course ◽

Cytosine Methylation ◽

Family Members ◽

Dynamic Network ◽

Dna Methyltransferases ◽

Genome Wide ◽

Distinct Sequence ◽

Adenosyl Methionine ◽

Methylation Patterns

ABSTRACTCytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT’s activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.

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Global Analysis of Pub1p Targets Reveals a Coordinate Control of Gene Expression through Modulation of Binding and Stability

Molecular and Cellular Biology ◽

10.1128/mcb.25.13.5499-5513.2005 ◽

2005 ◽

Vol 25 (13) ◽

pp. 5499-5513 ◽

Cited By ~ 51

Author(s):

Radharani Duttagupta ◽

Bin Tian ◽

Carol J. Wilusz ◽

Danny T. Khounh ◽

Patricia Soteropoulos ◽

...

Keyword(s):

Gene Expression ◽

Rna Binding ◽

Global Analysis ◽

Affinity Purification ◽

Mrna Turnover ◽

Sequence Motifs ◽

Posttranscriptional Control ◽

Control Of Gene Expression ◽

Transcript Stability ◽

Distinct Sequence

ABSTRACT Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1Δ strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1Δ strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1Δ strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3′ untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.

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PS2-021. Distinct sequence motifs in the HCV poly-U/UC RNA domain are recognized as non-self by RIG-I

Cytokine ◽

10.1016/j.cyto.2011.07.181 ◽

2011 ◽

Vol 56 (1) ◽

pp. 68-69

Author(s):

Gretja Schnell ◽

Michael Gale

Keyword(s):

Sequence Motifs ◽

Distinct Sequence

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Sensitive identification of known and unknown protease activities by unsupervised linear motif deconvolution

10.1101/2021.11.15.468703 ◽

2021 ◽

Author(s):

Anuli C Uzozie ◽

Theodore G Smith ◽

Siyuan Chen ◽

Philipp F Lange

Keyword(s):

Sequence Data ◽

Human Cell Line ◽

Sequence Motifs ◽

Web Interface ◽

Linear Motif ◽

Control Comparison ◽

Protein Sequence Data ◽

Distinct Sequence ◽

Urothelial Bladder ◽

Supervised Classification Methods

The cleavage-site specificities for many proteases are not well-understood, restricting the utility of supervised classification methods. We present an algorithm and web interface to overcome this limitation through the unsupervised detection of overrepresented patterns in protein sequence data, providing insight into the mixture of protease activities contributing to a complex system. Here, we apply the RObust LInear Motif Deconvolution (RoLiM) algorithm to confidently detect substrate cleavage patterns for SARS-CoV-2 Mpro protease in N terminome data of an infected human cell line. Using mass spectrometry-based peptide data from a case-control comparison of 341 primary urothelial bladder cancer cases and 110 controls, we identified distinct sequence motifs indicative of increased MMP activity in urine from cancer patients. Evaluation of N terminal peptides from patient plasma post-chemotherapy detected novel Granzyme B/Corin activity. RoLiM will enhance unbiased investigation of peptide sequences to establish the composition of known and uncharacterized protease activities in biological systems.

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