Optical Manipulation of Protein Activity and Protein Interactions Using Caged Proteins and Optical Switch Protein Conjugates

2011 ◽  
pp. 213-231
Author(s):  
Yuling Yan ◽  
Gerard Marriott
Keyword(s):  
Protein Interactions ◽  
Optical Switch ◽  
Optical Manipulation ◽  
Protein Activity ◽  
Protein Conjugates

scholarly journals Improvement of Capture Compound Mass Spectrometry Technology (CCMS) for the Profiling of Human Kinases by Combination with 2D LC-MS/MS

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Jenny J. Fischer ◽  
Olivia Graebner ◽  
Mathias Dreger ◽  
Mirko Glinski ◽  
Sabine Baumgart ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Small Molecule ◽  
Protein Interactions ◽  
Protein Identification ◽  
Spectrometric Analysis ◽  
Proteomic Profiling ◽  
Protein Conjugates ◽  
Input Material ◽  
Mass Spectrometry Technology

An increasingly popular and promising field in functional proteomics is the isolation of proteome subsets based on small molecule-protein interactions. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate captured protein conjugates from complex biological samples for direct protein identification by liquid chromatography/mass spectrometry (nLC-MS/MS). In this study we used staurosporine as a selectivity group for analysis in HepG2 cells derived from human liver. In the present study, we combined the functional isolation of kinases with different separation workflows of automated split-free nanoflow liquid chromatography prior to mass spectrometric analysis. Two different CCMS setups, CCMS technology combined with 1D LC-MS and 2D LC-MS, were compared regarding the total number of kinase identifications. By extending the chromatographic separation of the tryptic digested captured proteins from 1D LC linear gradients to 2D LC we were able to identify 97 kinases. This result is similar to the 1D LC setup we previously reported but this time 4 times less input material was needed. This makes CCMS of kinases an even more powerful tool for the proteomic profiling of this important protein family.

scholarly journals VarQ: a tool for the structural analysis of Human Protein Variants

2018 ◽  
Author(s):  
Leandro Radusky ◽  
Carlos Modenutti ◽  
Javier Delgado ◽  
Juan P. Bustamante ◽  
Sebastian Vishnopolska ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Single Amino Acid ◽  
Disease Development ◽  
Functional Effect ◽  
Single Nucleotide Variants ◽  
Protein Activity ◽  
Single Nucleotide ◽  
Online Tool ◽  
Protein Variants

AbstractUnderstanding the functional effect of Single Amino acid Substitutions (SAS), derived from the occurrence of single nucleotide variants (SNVs), and their relation to disease development is a major issue in clinical genomics. Even though there are several bioinformatic algorithms and servers that predict if a SAS can be pathogenic or not they give little or non-information on the actual effect on the protein function. Moreover, many of these algorithms are able to predict an effect that no necessarily translates directly into pathogenicity. VarQ Web Server is an online tool that given an UniProt id automatically analyzes known and user provided SAS for their effect on protein activity, folding, aggregation and protein interactions among others. VarQ assessment was performed over a set of previously manually curated variants, showing its ability to correctly predict the phenotypic outcome and its underlying cause. This resource is available online at http://varq.qb.fcen.uba.ar/.Contact: [email protected] Information & Tutorials may be found in the webpage of the tool.

scholarly journals Protein Hydroxylation by Hypoxia-Inducible Factor (HIF) Hydroxylases: Unique or Ubiquitous?

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 384 ◽  
Author(s):  
Moritz Strowitzki ◽  
Eoin Cummins ◽  
Cormac Taylor
Keyword(s):  
Protein Interactions ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Response ◽  
Protein Protein Interactions ◽  
Protein Activity ◽  
Prolyl Hydroxylases ◽  
Von Hippel Lindau ◽  
Technical Advances ◽  
And Migration ◽  
Α Subunits

All metazoans that utilize molecular oxygen (O2) for metabolic purposes have the capacity to adapt to hypoxia, the condition that arises when O2 demand exceeds supply. This is mediated through activation of the hypoxia-inducible factor (HIF) pathway. At physiological oxygen levels (normoxia), HIF-prolyl hydroxylases (PHDs) hydroxylate proline residues on HIF-α subunits leading to their destabilization by promoting ubiquitination by the von-Hippel Lindau (VHL) ubiquitin ligase and subsequent proteasomal degradation. HIF-α transactivation is also repressed in an O2-dependent way due to asparaginyl hydroxylation by the factor-inhibiting HIF (FIH). In hypoxia, the O2-dependent hydroxylation of HIF-α subunits by PHDs and FIH is reduced, resulting in HIF-α accumulation, dimerization with HIF-β and migration into the nucleus to induce an adaptive transcriptional response. Although HIFs are the canonical substrates for PHD- and FIH-mediated protein hydroxylation, increasing evidence indicates that these hydroxylases may also have alternative targets. In addition to PHD-conferred alterations in protein stability, there is now evidence that hydroxylation can affect protein activity and protein/protein interactions for alternative substrates. PHDs can be pharmacologically inhibited by a new class of drugs termed prolyl hydroxylase inhibitors which have recently been approved for the treatment of anemia associated with chronic kidney disease. The identification of alternative targets of HIF hydroxylases is important in order to fully elucidate the pharmacology of hydroxylase inhibitors (PHI). Despite significant technical advances, screening, detection and verification of alternative functional targets for PHDs and FIH remain challenging. In this review, we discuss recently proposed non-HIF targets for PHDs and FIH and provide an overview of the techniques used to identify these.

scholarly journals Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

1986 ◽  
Vol 6 (12) ◽  
pp. 4168-4178 ◽  
Author(s):  
C L Peterson ◽  
K Orth ◽  
K L Calame
Keyword(s):  
Heavy Chain ◽  
Protein Interactions ◽  
Binding Sites ◽  
Immunoglobulin Heavy Chain ◽  
Lymphoid Cells ◽  
Protein Protein Interactions ◽  
Protein Activity ◽  
Enhancer Element ◽  
Enhancer Sequences

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.

Post-translational modifications in T cells in systemic erythematosus lupus

Rheumatology ◽  
2021 ◽  
Author(s):  
Fan Yang ◽  
Jin Lin ◽  
Weiqian Chen
Keyword(s):  
T Cell ◽  
Signaling Pathways ◽  
Protein Interactions ◽  
B Lymphocyte ◽  
Cell Function ◽  
Protein Protein Interactions ◽  
Protein Activity ◽  
Post Translational Modifications ◽  
T Cell Function ◽  
B Lymphocyte Stimulator

Abstract Systemic erythematosus lupus (SLE) is a classic autoimmune disease characterized by multiple autoantibodies and immune-mediated tissue damage. The etiology of this disease is still unclear. A new drug, Belimumab, which acts against the B-lymphocyte stimulator (BLyS), can effectively improve the condition of SLE patients, but it cannot resolve all SLE symptoms. The discovery of novel, precise therapeutic targets is urgently needed. It is well known that abnormal T cell function is one of the most crucial factors contributing to the pathogenesis of SLE. Protein post-translational modifications (PTMs), including phosphorylation, glycosylation, acetylation, methylation, ubiquitination, and SUMOylation, have been emphasized for their roles in activating protein activity, maintaining structural stability, regulating protein-protein interactions and mediating signaling pathways, in addition to other biological functions. Summarizing the latest data in this area, this review focuses on the potential roles of diverse PTMs in regulating T cell function and signaling pathways in SLE pathogenesis, with the goal of identifying new targets for SLE therapy.

scholarly journals A Logical Molecular Circuit for Programmable and Autonomous Regulation of Protein Activity Using DNA Aptamer–Protein Interactions

2012 ◽  
Vol 134 (51) ◽  
pp. 20797-20804 ◽  
Author(s):  
Da Han ◽  
Zhi Zhu ◽  
Cuichen Wu ◽  
Lu Peng ◽  
Leiji Zhou ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Dna Aptamer ◽  
Protein Activity ◽  
Autonomous Regulation

scholarly journals Integrative Proteomic Profiling of Protein Activity and Interactions Using Protein Arrays

2012 ◽  
Vol 11 (11) ◽  
pp. 1167-1176 ◽  
Author(s):  
Se-Hui Jung ◽  
Kangseung Lee ◽  
Deok-Hoon Kong ◽  
Woo Jin Kim ◽  
Young-Myeong Kim ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transglutaminase 2 ◽  
Proteomic Profiling ◽  
Protein Activity ◽  
Protein Arrays ◽  
Protein Functions ◽  
Quantitative Validation ◽  
Interaction Map ◽  
On Chip ◽  
Related Proteins

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA

1986 ◽  
Vol 6 (12) ◽  
pp. 4168-4178
Author(s):  
C L Peterson ◽  
K Orth ◽  
K L Calame
Keyword(s):  
Heavy Chain ◽  
Protein Interactions ◽  
Binding Sites ◽  
Immunoglobulin Heavy Chain ◽  
Lymphoid Cells ◽  
Protein Protein Interactions ◽  
Protein Activity ◽  
Enhancer Element ◽  
Enhancer Sequences

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.

scholarly journals FICD activity and AMPylation remodelling modulate human neurogenesis

2019 ◽  
Author(s):  
Pavel Kielkowski ◽  
Isabel Y. Buchsbaum ◽  
Volker C. Kirsch ◽  
Nina C. Bach ◽  
Micha Drukker ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Protein Interactions ◽  
Posttranslational Modification ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Protein Protein Interactions ◽  
Protein Activity ◽  
Mature Neurons ◽  
A Cell

Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signalling, localisation or protein-protein interactions1. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity2. In human cells AMPylation has been exclusively studied with the FICD protein3–6. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodelling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.

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