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1.
Methods Mol Biol ; 2406: 281-317, 2022.
Article in English | MEDLINE | ID: mdl-35089564

ABSTRACT

Rapid preparation of proteins for functional and structural analysis is a major challenge both in academia and industry. The number potential targets continuously increases and many are difficult to express proteins which, when produced in bacteria, result in insoluble and/or misfolded recombinant proteins, protein aggregates, or unusable low protein yield. We focus here on the baculovirus expression vector system which is now commonly used for heterologous production of human targets. This chapter describes simple and cost-effective protocols that enable iterative cycles of construct design, expression screening and optimization of protein production. We detail time- and cost-effective methods for generation of baculoviruses by homologous recombination and titer evaluation. Handling of insect cell cultures and preparation of bacmid for cotransfection are also presented.


Subject(s)
Baculoviridae , Genetic Vectors , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Cell Culture Techniques , Genetic Vectors/genetics , Humans , Insecta/genetics , Insecta/metabolism , Recombinant Proteins/metabolism
2.
Mol Cell ; 75(3): 483-497.e9, 2019 08 08.
Article in English | MEDLINE | ID: mdl-31253574

ABSTRACT

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.


Subject(s)
BRCA1 Protein/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , Deubiquitinating Enzymes/genetics , Histone Chaperones/genetics , Neoplasms/genetics , Binding Sites/genetics , Carrier Proteins/genetics , Cell Nucleus/genetics , Cell Nucleus/immunology , Cytoplasm/genetics , Cytoplasm/immunology , DNA Breaks, Double-Stranded , DNA Repair/immunology , Deubiquitinating Enzymes/immunology , HeLa Cells , Humans , Immunity, Cellular/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Neoplasms/immunology , Nuclear Matrix-Associated Proteins/genetics , Protein Binding/genetics , Ubiquitin/genetics , Ubiquitin-Specific Proteases/genetics , Ubiquitination/genetics
3.
Science ; 362(6414)2018 11 02.
Article in English | MEDLINE | ID: mdl-30385546

ABSTRACT

The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase. We screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.


Subject(s)
CYS2-HIS2 Zinc Fingers , Lenalidomide/pharmacology , Peptide Hydrolases/metabolism , Proteolysis/drug effects , Thalidomide/analogs & derivatives , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effects , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , HEK293 Cells , Humans , Ikaros Transcription Factor/metabolism , Proteome/metabolism , Thalidomide/pharmacology
4.
Nature ; 531(7596): 598-603, 2016 Mar 31.
Article in English | MEDLINE | ID: mdl-27029275

ABSTRACT

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.


Subject(s)
Biocatalysis , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Peptide Hydrolases/metabolism , Peptide Hydrolases/ultrastructure , Allosteric Regulation , Apoproteins/chemistry , Apoproteins/metabolism , Apoproteins/ultrastructure , Binding Sites , COP9 Signalosome Complex , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Carrier Proteins/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , Cullin Proteins/chemistry , Cullin Proteins/metabolism , Cullin Proteins/ultrastructure , DNA Damage , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/ultrastructure , Humans , Kinetics , Models, Molecular , Multiprotein Complexes/chemistry , Peptide Hydrolases/chemistry , Protein Binding , Ubiquitination , Ubiquitins/metabolism
5.
Methods Mol Biol ; 1258: 181-205, 2015.
Article in English | MEDLINE | ID: mdl-25447865

ABSTRACT

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.


Subject(s)
Baculoviridae/genetics , Gene Expression/genetics , Genetic Vectors/genetics , Insecta/genetics , Recombinant Proteins/genetics , Animals , Humans , Transfection/methods
6.
Methods Mol Biol ; 1261: 91-114, 2015.
Article in English | MEDLINE | ID: mdl-25502195

ABSTRACT

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.


Subject(s)
Baculoviridae/genetics , Cloning, Molecular/methods , Insecta/virology , Multiprotein Complexes/biosynthesis , Animals , Baculoviridae/metabolism , Cell Culture Techniques , Cell Line , Genetic Vectors/genetics , Genetic Vectors/metabolism , Insecta/cytology , Insecta/genetics , Multiprotein Complexes/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
7.
Nat Struct Mol Biol ; 21(3): 261-8, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24509834

ABSTRACT

Repair of DNA double-strand breaks via homologous recombination can produce double Holliday junctions (dHJs) that require enzymatic separation. Topoisomerase IIIα (TopIIIα) together with RMI1 disentangles the final hemicatenane intermediate obtained once dHJs have converged. How binding of RMI1 to TopIIIα influences it to behave as a hemicatenane dissolvase, rather than as an enzyme that relaxes DNA topology, is unknown. Here, we present the crystal structure of human TopIIIα complexed to the first oligonucleotide-binding domain (OB fold) of RMI1. TopIII assumes a toroidal type 1A topoisomerase fold. RMI1 attaches to the edge of the gate in TopIIIα through which DNA passes. RMI1 projects a 23-residue loop into the TopIIIα gate, thereby influencing the dynamics of its opening and closing. Our results provide a mechanistic rationale for how RMI1 stabilizes TopIIIα-gate opening to enable dissolution and illustrate how binding partners modulate topoisomerase function.


Subject(s)
Carrier Proteins/metabolism , DNA Topoisomerases, Type I/metabolism , DNA, Cruciform/genetics , Nuclear Proteins/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , DNA Breaks, Double-Stranded , DNA, Single-Stranded/genetics , DNA-Binding Proteins , Gene Deletion , Humans , Models, Molecular , Molecular Sequence Data , Oligonucleotides/chemistry , Oligonucleotides/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Solubility
8.
Proc Natl Acad Sci U S A ; 110(8): E633-42, 2013 Feb 19.
Article in English | MEDLINE | ID: mdl-23382212

ABSTRACT

The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the "ARCH domain." By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair.


Subject(s)
DNA Repair , Mutation , Receptor Protein-Tyrosine Kinases/metabolism , Transcription Factor TFIIH/physiology , Transcription, Genetic , Xeroderma Pigmentosum Group D Protein/genetics , Cell Line , Chromatin Immunoprecipitation , Discoidin Domain Receptor 1 , Humans , Iron-Sulfur Proteins/metabolism , Models, Molecular , Reverse Transcriptase Polymerase Chain Reaction , Trichothiodystrophy Syndromes/genetics , Xeroderma Pigmentosum Group D Protein/chemistry , Xeroderma Pigmentosum Group D Protein/metabolism
9.
Anal Biochem ; 407(1): 34-43, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20624369

ABSTRACT

The baculovirus expression vector system is recognized as a powerful and versatile tool for producing large quantities of recombinant proteins that cannot be obtained in Escherichia coli. Here we report (i) the purification of the recombinant cyclin-dependent kinase (CDK)-activating kinase (CAK) complex, which includes CDK7, cyclin H, and MAT1 proteins, and (ii) the functional characterization of CAK together with a detailed analysis and mapping of the phosphorylation states and sites using mass spectrometry (MS). In vitro kinase assay showed that recombinant CAK is able to phosphorylate the cyclin-dependent kinase CDK2 implicated in cell cycle progression and the carboxy-terminal domain (CTD) of the eukaryotic RNA polymerase II. An original combination of MS techniques was used for the determination of the phosphorylation sites of each constitutive subunit at both protein and peptide levels. Liquid chromatography (LC)-MS analysis of intact proteins demonstrated that none of the CAK subunits was fully modified and that the phosphorylation pattern of recombinant CAK is extremely heterogeneous. Finally, matrix-assisted laser desorption/ionization (MALDI)-MS and nanoLC-tandem mass spectrometry (MS/MS) techniques were used for the analysis of the major phosphorylation sites of each subunit, showing that all correspond to Ser/Thr phosphorylation sites. Phosphorylations occurred on Ser164 and Thr170 residues of CDK7, Thr315 residue of cyclin H, and Ser279 residue of MAT1.


Subject(s)
Phosphoproteins/chemistry , Recombinant Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cell Cycle Proteins , Chromatography, High Pressure Liquid/methods , Cyclin H/genetics , Cyclin H/isolation & purification , Cyclin H/metabolism , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/isolation & purification , Cyclin-Dependent Kinases/metabolism , Molecular Sequence Data , Nanotechnology/methods , Phosphopeptides/analysis , Phosphorylation , RNA Polymerase II/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transcription Factors , Cyclin-Dependent Kinase-Activating Kinase
10.
Anal Biochem ; 385(2): 383-5, 2009 Feb 15.
Article in English | MEDLINE | ID: mdl-19061853

ABSTRACT

We report a set of baculovirus transfer vectors for parallel expression of proteins in fusion with a panel of affinity tags including GST, protein A, thioredoxin, CBP, and FLAG. This suite includes vectors to generate recombinant baculovirus by homologous recombination in insect cells or using the Bac-to-Bac technology. An application of the vector suite approach to the vitamin D receptor (VDR), a protein mainly expressed as inclusion bodies in Escherichia coli, is presented. We found that expression in fusion with GST and protein A provided an efficient compromise of excellent purification with acceptable yields and costs.


Subject(s)
Affinity Labels , Baculoviridae/genetics , Cloning, Molecular/methods , Genetic Vectors , Proteins/genetics , Animals , Cell Line , Escherichia coli/genetics , Inclusion Bodies/genetics , Insecta , Recombinant Fusion Proteins/genetics , Transfection
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