Current protocols in chemical biology最新文献_第5页

Single-Protein-Specific Redox Targeting in Live Mammalian Cells and C. elegans 活体哺乳动物细胞和秀丽隐杆线虫的单蛋白特异性氧化还原靶向

Current protocols in chemical biology Pub Date : 2018-08-07 DOI: 10.1002/cpch.43

Alexandra Van Hall-Beauvais, Yi Zhao, Daniel A. Urul, Marcus J. C. Long, Yimon Aye

{"title":"Single-Protein-Specific Redox Targeting in Live Mammalian Cells and C. elegans","authors":"Alexandra Van Hall-Beauvais, Yi Zhao, Daniel A. Urul, Marcus J. C. Long, Yimon Aye","doi":"10.1002/cpch.43","DOIUrl":"10.1002/cpch.43","url":null,"abstract":"T-REX (targetable reactive electrophiles and oxidants) enables electrophile targeting in living systems with high spatiotemporal precision and at single-protein-target resolution. T-REX allows functional consequences of individual electrophile signaling events to be directly linked to on-target modifications. T-REX is accomplished by expressing a HaloTagged protein of interest (POI) and introducing a Halo-targetable bioinert photocaged precursor to a reactive electrophilic signal (RES). Light exposure releases the unfettered RES on demand, enabling precision modification of the POI due to proximity. Using alkyne-functionalized 4-hydroxynonenal (HNE) as a representative RES, this protocol delineates optimized strategies to (1) execute T-REX in live human cells and C. elegans, (2) quantitate the POI's RES-sensitivity by either azido-fluorescent-dye conjugation or (3) enrich using biotin-azide/streptavidin pulldown procedure in both model systems, and (4) identify the site of RES-labeling on the POI using proteomics. Built-in T-REX controls that allow users to directly confirm on-target/on-site specificity of RES-sensing are also described. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.43","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36376026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Monitoring PARKIN RBR Ubiquitin Ligase Activation States with UbFluor 用UbFluor监测PARKIN RBR泛素连接酶激活状态

Current protocols in chemical biology Pub Date : 2018-07-31 DOI: 10.1002/cpch.45

Peter K. Foote, Alexander V. Statsyuk

引用次数: 5

Metabolic Labeling of Prenylated Proteins Using Alkyne-Modified Isoprenoid Analogues 炔修饰类异戊二烯类似物对戊烯酰化蛋白的代谢标记

Current protocols in chemical biology Pub Date : 2018-07-30 DOI: 10.1002/cpch.46

Kiall F. Suazo, Alexander K. Hurben, Kevin Liu, Feng Xu, Pa Thao, Ch. Sudheer, Ling Li, Mark D. Distefano

{"title":"Metabolic Labeling of Prenylated Proteins Using Alkyne-Modified Isoprenoid Analogues","authors":"Kiall F. Suazo, Alexander K. Hurben, Kevin Liu, Feng Xu, Pa Thao, Ch. Sudheer, Ling Li, Mark D. Distefano","doi":"10.1002/cpch.46","DOIUrl":"10.1002/cpch.46","url":null,"abstract":"Protein prenylation involves the attachment of a farnesyl or geranylgeranyl group onto a cysteine residue located near the C-terminus of a protein, recognized via a specific prenylation motif, and results in the formation of a thioether bond. To identify putative prenylated proteins and investigate changes in their levels of expression, metabolic labeling and subsequent bioorthogonal labeling has become one of the methods of choice. In that strategy, synthetic analogues of biosynthetic precursors for post-translational modification bearing bioorthogonal functionality are added to the growth medium from which they enter cells and become incorporated into proteins. Subsequently, the cells are lysed and proteins bearing the analogues are then covalently modified using selective chemical reagents that react via bioorthogonal processes, allowing a variety of probes for visualization or enrichment to be attached for subsequent analysis. Here, we describe protocols for synthesizing several different isoprenoid analogues and describe how they are metabolically incorporated into mammalian cells, and the incorporation into prenylated proteins visualized via in-gel fluorescence analysis. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.46","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36354052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 15

XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases 工程嗜热聚合酶的XNA合成与反转录

Current protocols in chemical biology Pub Date : 2018-07-24 DOI: 10.1002/cpch.47

Christopher Cozens, Vitor B. Pinheiro

引用次数: 6

Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach 利用蛋白质组学肽库(ProPeL)方法表征蛋白激酶底物特异性

Current protocols in chemical biology Pub Date : 2018-06-19 DOI: 10.1002/cpch.38

Joshua M. Lubner, Jeremy L. Balsbaugh, George M. Church, Michael F. Chou, Daniel Schwartz

引用次数: 9

Generating Conformation-Specific Polyclonal and Monoclonal Anti-Protein Kinase C Antibodies and Anti–Active State Specific PKC Antibodies 生成构象特异性多克隆和单克隆抗蛋白激酶C抗体和抗活性状态特异性PKC抗体

Current protocols in chemical biology Pub Date : 2018-06-12 DOI: 10.1002/cpch.42

Darlene A. Pena, Denise M. V. Pacheco, Paulo S. L. Oliveira, Maria J. M. Alves, Deborah Schechtman

{"title":"Generating Conformation-Specific Polyclonal and Monoclonal Anti-Protein Kinase C Antibodies and Anti–Active State Specific PKC Antibodies","authors":"Darlene A. Pena, Denise M. V. Pacheco, Paulo S. L. Oliveira, Maria J. M. Alves, Deborah Schechtman","doi":"10.1002/cpch.42","DOIUrl":"10.1002/cpch.42","url":null,"abstract":"The protein kinase C (PKC) family of serine/ threonine kinases has been shown to play active roles as either suppressors or promoters of carcinogenesis in different types of tumors. Using antibodies that preferentially recognize the active conformation of classical PKCs (cPKCs), we have previously shown that in breast cancer samples the expression levels of cPKCs were similar in estrogen receptor–positive (ER+) as compared to triple-negative tumors; however, the levels of active cPKCs were different. Determining the activation status of PKCs and other kinases in tumors may thus aid therapeutic decisions. Further, in basic science these tools may be used to understand the spatial and temporal dynamics of PKC signaling under different stimuli and for co-immunoprecipitation studies to detect binding partners and substrates of active cPKCs. In this article, we describe how monoclonal and polyclonal anti–active state PKC antibodies can be obtained using rational approaches to select bona fide epitopes through inspection of the crystal structure of classical PKCs coupled to molecular modeling studies. We believe that this methodology can be used for other kinases and multi-domain enzymes that undergo changes in their conformation upon activation. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.42","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36244160","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generating FN3-Based Affinity Reagents Through Phage Display 通过噬菌体展示生成基于fn3的亲和试剂

Current protocols in chemical biology Pub Date : 2018-06-07 DOI: 10.1002/cpch.39

Kevin Gorman, Jennifer McGinnis, Brian Kay

引用次数: 3

Glycocalyx Scaffolding to Control Cell Surface Glycan Displays 控制细胞表面聚糖显示的糖萼支架

Current protocols in chemical biology Pub Date : 2018-06-05 DOI: 10.1002/cpch.40

Mia L. Huang, Ember M. Tota, Stephen Verespy III, Kamil Godula

{"title":"Glycocalyx Scaffolding to Control Cell Surface Glycan Displays","authors":"Mia L. Huang, Ember M. Tota, Stephen Verespy III, Kamil Godula","doi":"10.1002/cpch.40","DOIUrl":"10.1002/cpch.40","url":null,"abstract":"This article describes a protocol for remodeling cells with synthetic glycoprotein and glycolipid mimetics that are functionalized with lipid anchors, allowing for cell surface display of specific glycan structures in predefined nanoscale arrangements. The complex chemical heterogeneity of glycans found on the cell surface or the glycocalyx renders analysis of the individual contributions of glycans difficult. This technique allows for the precise study of individual glycans at different regions of the glycocalyx, and may be useful for interrogating glycan interactions in infection or immunity or in stem cell differentiation. CHO-Lec2 cells are prepared as adherent monolayers and, after reaching confluence, are incubated with the glycomaterials. Synthetic glycopolymers bearing α-2,3-sialyllactose glycans are used to decorate cellular surfaces in the form of 3D multivalent ligands projecting away from the cell surface, while α-2,6-sialyllactose glycolipid conjugates are used to anchor glycans in dynamic 2D arrays proximal to the cell membrane. Following washing, mimetic incorporation and glycan display can be analyzed using lectins with specificity for α-2,3- or α-2,6-linked sialic acids. Flow cytometry data reveals that cell surface remodeling with either glycoconjugate mimetic occurs efficiently in a dose-dependent manner. Combinations of glycoconjugates can also be employed simultaneously to generate a mixed glycocalyx with tunable composition and organization. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.40","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36243854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Identification of Phosphorylated Proteins on a Global Scale 全球范围内磷酸化蛋白的鉴定

Current protocols in chemical biology Pub Date : 2018-05-31 DOI: 10.1002/cpch.48

Anton Iliuk

引用次数: 1

Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase 利用Phi29 DNA聚合酶合成异种核酸(XNA)

Current protocols in chemical biology Pub Date : 2018-05-18 DOI: 10.1002/cpch.41

Leticia L. Torres, Vitor B. Pinheiro

引用次数: 14