Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types

ABSTRACT Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αβ) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.

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Proteomics: current techniques and potential applications to lung disease

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00301.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. L1-L23 ◽

Cited By ~ 70

Author(s):

Jan Hirsch ◽

Kirk C. Hansen ◽

Alma L. Burlingame ◽

Michael A. Matthay

Keyword(s):

Posttranslational Modifications ◽

Protein Separation ◽

Protein Identification ◽

Cell Types ◽

Protein Profiling ◽

Complex Samples ◽

Potential Value ◽

Potential Applications ◽

Tissue Specimens ◽

Different Cell Types

Proteomics aims to study the whole protein content of a biological sample in one set of experiments. Such an approach has the potential value to acquire an understanding of the complex responses of an organism to a stimulus. The large vascular and air space surface area of the lung expose it to a multitude of stimuli that can trigger a variety of responses by many different cell types. This complexity makes the lung a promising, but also challenging, target for proteomics. Important steps made in the last decade have increased the potential value of the results of proteomics studies for the clinical scientist. Advances in protein separation and staining techniques have improved protein identification to include the least abundant proteins. The evolution in mass spectrometry has led to the identification of a large part of the proteins of interest rather than just describing changes in patterns of protein spots. Protein profiling techniques allow the rapid comparison of complex samples and the direct investigation of tissue specimens. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. These methodologies have made the application of proteomics on the study of specific diseases or biological processes under clinically relevant conditions possible. The quantity of data that is acquired with these new techniques places new challenges on data processing and analysis. This article provides a brief review of the most promising proteomics methods and some of their applications to pulmonary research.

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An ultrasensitive fiveplex activity assay for cellular kinases

Scientific Reports ◽

10.1038/s41598-019-55998-8 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Christian M. Smolko ◽

Kevin A. Janes

Keyword(s):

Protein Kinase ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Large Scale ◽

Cell Types ◽

Mitogen Activated Protein Kinase ◽

Activity Measurement ◽

Kinase Substrate ◽

Iκb Kinase ◽

Mitogen Activated Protein

AbstractProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

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Posttranslational Modifications Required for Cell Surface Localization and Function of the Fungal Adhesin Aga1p

Eukaryotic Cell ◽

10.1128/ec.2.5.1099-1114.2003 ◽

2003 ◽

Vol 2 (5) ◽

pp. 1099-1114 ◽

Cited By ~ 13

Author(s):

Guohong Huang ◽

Mingliang Zhang ◽

Scott E. Erdman

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Posttranslational Modifications ◽

Transmembrane Domain ◽

Signal Sequence ◽

Cell Types ◽

Fungal Species ◽

Surface Proteins ◽

Functional Requirements

ABSTRACT Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.

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ETO-2 Associates with SCL in Erythroid Cells and Megakaryocytes and Provides Repressor Functions in Erythropoiesis

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10235-10250.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10235-10250 ◽

Cited By ~ 101

Author(s):

Anna H. Schuh ◽

Alex J. Tipping ◽

Allison J. Clark ◽

Isla Hamlett ◽

Boris Guyot ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Protein Complexes ◽

Fetal Liver ◽

Regulation Of Gene Expression ◽

Erythroid Differentiation ◽

Cell Types ◽

Erythroid Cells ◽

Proteomic Approach ◽

Endogenous Proteins

ABSTRACT Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

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Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

10.1101/2022.01.13.476100 ◽

2022 ◽

Author(s):

Lewis A Macdonald ◽

Gillian C A Taylor ◽

Jennifer M Brisbane ◽

Ersi Christodoulou ◽

Lucy Scott ◽

...

Keyword(s):

Mouse Models ◽

Protein Function ◽

Mammalian Cells ◽

Degradation Kinetics ◽

Cell Types ◽

Mouse Genetics ◽

Chemical Genetic ◽

Endogenous Proteins ◽

Specific Degradation

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II, two essential regulators of mitotic chromosome structure, revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

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Activity-Dependent Probes Containing Epsilon-N-Thioacyllysine and -Epsilon-N-Acyl-(Delta-Aza)lysine Residues

10.26434/chemrxiv.10315502.v1 ◽

2019 ◽

Author(s):

Michael Bæk, ◽

Pablo Martín-Gago ◽

Jonas S. Laursen ◽

Julie L. H. Madsen ◽

Saswati Chakladar ◽

...

Keyword(s):

Posttranslational Modifications ◽

Protein Function ◽

Splice Variants ◽

Peptide Sequence ◽

Fluorescence Measurements ◽

Lysine Residues ◽

Covalent Adducts ◽

Endogenous Proteins ◽

Activity Dependent ◽

Hydrolysis Of

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. Here, we describe development of peptide activity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of epsilon-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the epsilon-N-glutaryllysine and epsilon-N-myristoyllysine analogues, were successfully applied to enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

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Activity-Dependent Probes Containing Epsilon-N-Thioacyllysine and -Epsilon-N-Acyl-(Delta-Aza)lysine Residues

10.26434/chemrxiv.10315502 ◽

2019 ◽

Author(s):

Michael Bæk, ◽

Pablo Martín-Gago ◽

Jonas S. Laursen ◽

Julie L. H. Madsen ◽

Saswati Chakladar ◽

...

Keyword(s):

Posttranslational Modifications ◽

Protein Function ◽

Splice Variants ◽

Peptide Sequence ◽

Fluorescence Measurements ◽

Lysine Residues ◽

Covalent Adducts ◽

Endogenous Proteins ◽

Activity Dependent ◽

Hydrolysis Of

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. Here, we describe development of peptide activity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of epsilon-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the epsilon-N-glutaryllysine and epsilon-N-myristoyllysine analogues, were successfully applied to enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

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Multiplexed Imaging of Posttranslational Modifications of Endogenous Proteins in Live Cells

Methods in Molecular Biology - Multiplexed Imaging ◽

10.1007/978-1-0716-1593-5_3 ◽

2021 ◽

pp. 31-41

Author(s):

Yuko Sato ◽

Hiroshi Kimura

Keyword(s):

Posttranslational Modifications ◽

Live Cells ◽

Multiplexed Imaging ◽

Endogenous Proteins

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Toll-Like Receptors, Their Ligands, and Atherosclerosis

The Scientific World JOURNAL ◽

10.1100/tsw.2011.36 ◽

2011 ◽

Vol 11 ◽

pp. 437-453 ◽

Cited By ~ 21

Author(s):

Conrad P. Hodgkinson ◽

Shu Ye

Keyword(s):

Arterial Wall ◽

Cell Types ◽

Innate Immune ◽

Toll Like Receptors ◽

Increased Risk ◽

Inflammatory Proteins ◽

Endogenous Proteins ◽

Molecular Patterns ◽

Microbial Agents

Atherosclerosis is a disease characterized by inflammation in the arterial wall. Atherogenesis is dependent on the innate immune response involving activation of Toll-like receptors (TLRs) and the expression of inflammatory proteins. TLRs, which recognize various pathogen-associated molecular patterns, are expressed in various cell types within the atherosclerotic plaque. Microbial agents are associated with an increased risk of atherosclerosis and this is, in part, due to activation of TLRs. Recently considerable evidence has been provided suggesting that endogenous proteins promote atherosclerosis by binding to TLRs. In this review, we describe the role of TLRs in atherosclerosis with particular emphasis on those atherogenic endogenous proteins that have been implicated as TLR ligands.

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