Competent BL21 Research Articles

Expression and Purification of Human U1-70K (snRNP70) and its BAD Domains Using an E. coli Expression System.

U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.

Recombinant Antimicrobial Peptide Fusion Between Crotalicidin Fragment Tag and Bacteriophage Endolysin T5 as a Potential Antibacterial Agent Against Multidrug Resistant Gram-Negative Bacteria: A Research Protocol

Introduction: Endolysins, antimicrobial peptides that disrupt the peptidoglycan (PG) layer of bacteria, are a strong alternative to common antibiotics and less prone to antibiotic resistance. However, endolysins are ineffective against gram-negative bacteria due to an additional outer membrane (OM) blocking the PG layer. This research protocol aims to address this caveat by developing a novel recombinant endolysin peptide, EndoT5-Ctn (15-34), comprised of phage Endolysin T5 and a Crotalicidin tag (Ctn (15-34)) capable of permeabilizing the OM. This would render the endolysin effective against gram-negative bacteria such as multidrug-resistant (MDR) Pseudomonas aeruginosa. Methods: Experimentation will begin with recombinant DNA techniques to engineer a bacterial vector which expresses the EndoT5-Ctn (15-34) peptide. Protein expression will be achieved in competent BL21 (DE3) E. coli strains before protein isolation with lysis methods and immobilized-metal affinity chromatography. Resulting sample concentrations will be assayed using spectrophotometry. Finally, in vivo minimal inhibitory and bactericidal concentration assays will be conducted on MDR P. aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) to evaluate bactericidal efficacy. Results: A minimal bactericidal concentration of EndoT5-Ctn (15-34) recombinant endolysin is expected to yield significant bactericidal activity against the MDR P. aeruginosa strain with limited effect on the gram-positive MRSA. A higher concentration of recombinant endolysin is expected to maintain its gram-negative bactericidal efficacy with an imported gram-positive bactericidal efficacy. Discussion: The observed bactericidal efficacy on AMR and non-AMR strains on P. aeruginosa will prompt further exploration of the therapeutic feasibility of this therapy regarding factors such as cytotoxicity, immunogenicity, and thermal stability. The potential introduction of recombinant endolysins presents an alternative treatment for infections with reduced risk of inducing resistant mutations. Conclusion: The EndoT5-Ctn (15-34) recombinant endolysin is a novel anti-bacterial agent which is expected to have significant bactericidal effects on MDR strains of gram-negative bacteria which current antibiotics have been ineffective at combating.

Construction of the Five-point Mutant in Novel Aspartokinase and Its Enzymatic Characterization from Corynebacterium pekinense

Purpose: To improve the activity of the first key allosteric enzyme-aspartate kinase (AK) in the aspartate amino acid synthesis pathway, and to weaken or remove the terminal products lysine (Lys) and threonine (Thr) synergistic feed back inhibition of AK. Methods: On this basis of the four-point mutant T379N/A380C/G171I/Y198N AK(NCIN AK) of Corynebacterium pekinense, it was found that the highly conserved mutation sites of Gly295 was bound with inhibitor Thr by hydrogen bonds. The site of Gly295 were selected for site-directed mutation. The plasmid was extracted and transformed into E. coli competent BL21 to induce expression. High-throughput screening was used to obtain mutant strains with significantly improved enzyme activity. Enzyme kinetic study and characterization of enzyme properties were performed on the wild type(WT) and the five-point mutant T379N/A380C/G171I/Y198N/G295L AK(NCINL AK). Results: The five-point mutant NCINL AK was successfully constructed, and its maximum reaction rate Vmax was 259 U/(mg·min). The Km value of the Michaelis constant of the mutant reduced from 3.44 mmol/L to 0.93 mmol/L, and the substrate affinity was enhanced compared with the wild type enzyme. The hill coefficient n value was reduced from 2.73 to 1.21, and the positive synergy was decreased. The optimum temperature of NCINL AK increased from 25 ℃ to 28 ℃, the optimum pH value increased from 8.0 to 8.5, and the half-life was extended from 4.7 h to 5.2 h. Compared with the wild type WT AK, the five-point mutant NCINL AK had different inhibitory effects at different substrate inhibitors with concentration of 0.2, 1.0, 5.0, 10.0 mmol/L and even the inhibitory effect of Lys was completely lifted, and it had an activation effect under the condition of 10 mmol/L Thr. Conclusion: In this experiment, the five-point mutant NCINL AK with enzyme activity increased by 87.20 times was obtained, and the enzymatic properties were also significantly improved. Within a certain inhibitor concentration range, the feedback-inhibition effect of Lys on AK was basically lifted, and to a certain extent releasing Thr's feedback inhibition provided the possibility for the accumulation of large amounts of aspartate amino acids and provided reference for constructing high-yield aspartate amino acid strains. It was possible for us to get the pollution-free and high-efficiency amino acids such as methionine (Met).

Recombinant expression and purification of adenocarcinoma GPR161 receptor

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer and very few therapeutic options are currently available for its treatment. Interestingly, G-protein coupled receptor 161 (GPR161) is expressed in TNBC cells and can activate the mammalian target of the rapamycin complex 1 signaling pathway. GPR161 and Ras GTPase-activating-like protein, a protein involved in intracellular signaling, proliferation, and cellular adhesion, have been shown to genetically interact in human breast cancer cells. Targeting of GPR161 by monoclonal antibodies may therefore be a strategy to develop diagnostics and therapeutics for TNBC. Thus, to obtain such monoclonal antibodies, we synthesized the GPR161 gene de novo, cloned it into the pET32 expression plasmid, and used the recombinant plasmid to transform the competent BL21 (DE3) strain of Escherichia coli. The recombinant GPR161 gene was designed to contain an N-terminal thioredoxin tag, a thrombin site, the GPR161 sequence, and a C-terminalhexa-histidine tag to facilitate purification by metal-affinity chromatography. Following purification of the recombinant GPR161 (rGPR161) protein using a HisTrap column, we characterized the protein by Western blotting and mass spectrometry. The rGPR161 protein had a molecular mass of ~49 kDa and its identity as rGPR161 was confirmed by mass spectrometry data using the SwissProt database and the Mascot program. Future studies will involve the development of monoclonal antibodies using rGPR161 as the immunogen.

Optimization of Cloning Conditions for High-level Production of Recombinant Mouse Interleukin-2 in Escherichia coli

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High-level Expression of Tetanus Toxin Fragment C in Escherichia coli.

Fragment C is the C-terminal domain of the heavy chain of tetanus toxin that can promote the immune response against the lethal dose of this toxin. Therefore, this portion can be considered as a candidate vaccine against tetanus infection, which occurs by Clostridium tetani. The present study aimed to compare the expression of tetanus toxin fragment C in Escherichia coli BL21 (DE3) pLysS cells having a high tolerance to toxins between two different expression vectors, namely pET22b and pET28a, using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. After DNA extraction from Harvard CN49205 strain of C. tetani, the gene of interest was amplified using polymerase chain reaction, and then sequenced and cloned into the expression vectors of pET22b and pET28a, transformed into competent BL21 (DE3) pLysS cells, and finally expressed using an optimized protocol. The cells were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at four different incubation temperatures (i.e., 37, 33, 30, and 25 °C) and three different incubation times (i.e., 1, 2, and 3 h). Although the SDS-PAGE and western blot analyses confirmed the expression of the recombinant fragment C (r-fragment C) ligated into both of the expression vectors, pET28a showed a higher r-fragment C expression level than the other vector (38.66 mg/L versus 32.33 mg/L, P<0.05). An optimal expression condition was acquired 3 h after 1 mM IPTG induction at 25 °C. The results demonstrated that E. coli BL21 (DE3) pLysS as an expression host in combination with pET-28a as an expression vector was a more compatible expression system to express the fragment C of tetanus toxin, compared to E. coli BL21 (DE3) pLysS/pET-22b expression system. Overall, these results may represent an opportunity to improve the expression system for the production of tetanus toxin vaccine using recombinant protein strategy.

Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate-specific membrane antigen.

The present study aimed to identify, express and purify an immunogenic fragment in the ectodomain of prostate-specific membrane antigen (PSMA) within a fusion protein. The PSMA amino acid sequence published in National Center for Biotechnology Information GenBank was used to determine sequence homology and immunogenic index analyses, additionally using BLASTN, Protean and ExPASy software to predict the polypeptide sequences of immunogenic epitopes. The gene sequence encoding the ectodomain of the polypeptide immunogenic fragments, containing the identified immunogenic epitopes, was generated using whole-gene synthesis. Prokaryotic expression vector pET-32a-r-ectodomain-PSMA was constructed and the recombinant plasmids were transformed into competent BL21 (DE3) Escherichia coli, which was followed by induction of recombinant protein expression using isopropyl-β-D-thiogalactopyranoside. Fusion proteins were isolated and purified using affinity chromatography and their immune activity was subsequently investigated using western blot analysis. Purified protein was used to immunize BALB/c mice in order to generate polyclonal antibodies, and the binding of polyclonal antibodies to prostate cancer cell lines in vitro was evaluated using flow cytometry. A total of 3 polypeptide fragments with high specificity were identified following analysis using numerous software packages, and the gene sequences encoding regions containing the 2 most immunogenic fragments were synthesized and successfully inserted into the prokaryotic expression vector pET-32a-r-ectodomain-PSMA. The recombinant PSMA protein fragment had a molecular weight of ~50 kDa and 95% purity. Western blot analysis revealed that the r-ectodomain-PSMA fusion protein specifically bound to the anti-PSMA ectodomain monoclonal antibody. Flow cytometry demonstrated that polyclonal antibodies raised against these recombinant proteins could specifically bind to PSMA-positive LNCaP cells, but not to PSMA-negative PC-3 cells. An immunogenic fragment in the ectodomain of PSMA was successfully expressed and purified. The present study, therefore, provides a basis for the preparation of an anti-PSMA small humanized monoclonal antibody.

120 IDENTIFICATION OF THE RELATIVELY PREDOMINANT BUFFALO INTERFERON tau ISOFORM AND ITS EXPRESSION IN ESCHERICHIA COLI

The maternal pregnancy recognition factor interferon tau (IFN-τ) is expressed in multiple isoforms in all pecoran ruminant species. Interferon-τ, as the first pregnancy signaling molecule, performs a significant role in implantation as well as establishment of pregnancy. Due to low reproductive efficiency of buffalo compared with bovine and IFN-τ being the key molecule of reproductive physiology in ruminants, the objective of our study was framed to identify the various IFN-τ transcripts in buffalo embryonic trophoblast cells, to know their relative abundance to identify the relatively predominant isoform, and lastly to clone and express it in a heterologous host. Following total cellular RNA extraction from primary trophectodermal cells, RT-PCR was performed using gene-specific primers designed against known bovine IFN-τ sequence. Cloning of the amplified product and screening of the recombinant colonies gave 13 distinct cDNA variants that encoded for 8 distinct buffalo IFN-τ isoforms. These buffalo IFN-τ isoforms have a greater nucleotide and amino acid homology with caprine IFN-τ (98–100% and 96–100%) than ovine (94–97% and 90–95%) and bovine (89.6–90.6% and 82–86%), respectively. The novel buffalo IFN-τ isoforms showed pronounced nucleotide and amino acid sequence identity with one another (99.1–99.8% and 98–99%) but only moderate identity with previously identified buffalo IFN-τ (90–92% and 82–86%). All the 13 transcript sequences were accepted in GenBank. Out of 8 isoforms, buffalo IFN-τ1 has been found to be the relatively predominant, which was subcloned into expression vector pET 22b without signal sequence from pJET cloning vector and expressed in competent BL21 (DE3) Escherichia coli strain. Expression of the recombinant protein in soluble form was induced by isopropyl β-D-1-thiogalactopyranoside (0.1 mM) at 30°C for 6 h. The recombinant BuIFN-τ obtained was confirmed by Western blot using anti-HIS antibody. A new 20-kDa protein was detected coinciding the molecular weight of IFN-τ reported earlier in literature. In conclusion, the current study revealed that there are 8 different isoforms of IFN-τ that are expressed in trophectodermal out-growths during early pregnancy of buffalo. Predominantly found isoform IFN-τ 1 was expressed in pET 22b vector, and recombinant soluble protein was confirmed by Western blot.

Expression and affinity purification of recombinant human epidermal growth factor receptor-2 affibody with C-terminal cystein

To prepare the modified ZHER2V2 affibody with amino-terminal HEHEHE sequence and carboxyl-terminal GGGC sequence by gene recombinant expression,which is the basis for invasive HER2 imaging with affibody. The encoded affibody gene was optimized by codon preference of E. coli with gene designer software. The N-terminal of affibody was fused with HEHEHE sequence,while the C-terminal was fused with GGGC sequence. The synthetic gene was confirmed by Hind 3 endonuclease restriction and gene sequencing. The human epidermal growth factor receptor-2(HER2)affibody gene was sub-cloned into pET22b(+)plasmid and transformed into competent BL21(DE3)bacteria. The expression of modified affibody was induced with isopropyl Β-D-1-thiogalactopyranoside(IPTG)and identified by SDS-PAGE. The affibody was purified by nickel affinity binding and imidazole elution. The purified affibody was labeled with (68)Ga and its affinity was determined by saturation analysis with HER2-positive cells MDA-MB-361. The affibody gene containing N-terminal HEHEHE and C-terminal GGGC sequences were confirmed by Hind 3 endonuclease restriction and gene sequencing. A newly expressed 8×10(3) protein was expressed from the induced recombinant bacteria identified by SDS-PAGE after sub-cloning HER2 affibody gene into pET22b(+)plasmid,transforming recombinant plasmid into competent BL21(DE3)bacteria and inducing the recombinant bacteria with IPTG. The expressed protein was purified from nickel agarose by 60 mmol/L imidazole eluting. The affinity Kd value of (68)Ga labeled affibody to HER2 positive MDA-MB-361 cells was 1.5 nmol/L. The affiibody ZHER2V2 containing N-terminal HEHEHE and C-terminal GGGC was successfully prepared by gene optimization,recombinant expression and affinity purification.

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Several gram-positive pathogenic bacteria employ near-iron transporter (NEAT) domains to acquire heme from hemoglobin during infection. However, the structural requirements and mechanism of action for NEAT-mediated heme extraction remains unknown. Bacillus anthracis exhibits a rapid growth rate during systemic infection, suggesting that the bacterium expresses efficient iron acquisition systems. To understand how B. anthracis acquires iron from heme sources, which account for 80% of mammalian iron stores, we investigated the properties of the five-NEAT domain hemophore IsdX2. Using a combination of bioinformatics and site-directed mutagenesis, we determined that the heme extraction properties of IsdX2 are dependent on an amino acid with an amide side chain within the 310-helix of the NEAT domain. Additionally, we used a spectroscopic analysis to show that IsdX2 NEAT domains only scavenge heme from methemoglobin (metHb) and that autoxidation of oxyhemoglobin to metHb must occur prior to extraction. We also report the crystal structures of NEAT5 wild type and a Q29T mutant and present surface plasmon resonance data that indicate that the loss of this amide side chain reduces the affinity of the NEAT domain for metHb. We propose a model whereby the amide side chain is first required to drive an interaction with metHb that destabilizes heme, which is subsequently extracted and coordinated in the aliphatic heme-binding environment of the NEAT domain. Because an amino acid with an amide side chain in this position is observed in NEAT domains of several genera of gram-positive pathogenic bacteria, these results suggest that specific targeting of this or nearby residues may be an entry point for inhibitor development aimed at blocking bacterial iron acquisition during infection.

Prodrug Activation by Cryptosporidium Thymidine Kinase

Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr805, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.

A Novel Interaction Linking the FAS-II and Phthiocerol Dimycocerosate (PDIM) Biosynthetic Pathways

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.

The Carotenoid Cleavage Dioxygenase 1 Enzyme Has Broad Substrate Specificity, Cleaving Multiple Carotenoids at Two Different Bond Positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').

Phosphorylation-dependent Binding of Cyclin B1 to a Cdc6-like Domain of Human Separase

Sister chromatids are held together by the ring-shaped cohesin complex, which likely entraps both DNA-double strands in its middle. This tie is resolved in anaphase when separase, a giant protease, becomes active and cleaves the kleisin subunit of cohesin. Premature activation of separase and, hence, chromosome missegregation are prevented by at least two inhibitory mechanisms. Although securin has long been appreciated as a direct inhibitor of separase, surprisingly its loss has basically no phenotype in mammals. Phosphorylation-dependent binding of Cdk1 constitutes an alternative way to inhibit vertebrate separase. Its importance is illustrated by the premature loss of cohesion when Cdk1-resistant separase is expressed in mammalian cells without or with limiting amounts of securin. Here, we demonstrate that crucial inhibitory phosphorylations occur within a region of human separase that is also shown to make direct contact with the cyclin B1 subunit of Cdk1. This region exhibits a weak homology to Saccharomyces cerevisiae Cdc6 of similar Cdk1 binding behavior, thereby establishing phosphoserine/threonine-mediated binding of partners as a conserved characteristic of B-type cyclins. In contrast to the Cdc6-like domain, the previously identified serine 1126 phosphorylation is fully dispensable for Cdk1 binding to separase fragments. This suggests that despite its in vivo relevance, it promotes complex formation indirectly, possibly by inducing a conformational change in full-length separase.

Characterization of the Cytochrome c Oxidase Assembly Factor Cox19 of Saccharomyces cerevisiae

Cox19 is an important accessory protein in the assembly of cytochrome c oxidase in yeast. The protein is functional when tethered to the mitochondrial inner membrane, suggesting its functional role within the intermembrane space. Cox19 resembles Cox17 in having a twin CX(9)C sequence motif that adopts a helical hairpin in Cox17. The function of Cox17 appears to be a Cu(I) donor protein in the assembly of the copper centers in cytochrome c oxidase. Cox19 also resembles Cox17 in its ability to coordinate Cu(I). Recombinant Cox19 binds 1 mol eq of Cu(I) per monomer and exists as a dimeric protein. Cox19 isolated from the mitochondrial intermembrane space contains variable quantities of copper, suggesting that Cu(I) binding may be a transient property. Cysteinyl residues important for Cu(I) binding are also shown to be important for the in vivo function of Cox19. Thus, a correlation exists in the ability to bind Cu(I) and in vivo function.

Mutational Analysis of the Catalytic Domain of O-Linked N-Acetylglucosaminyl Transferase

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.

A Proteomic Microarray Approach for Exploring Ligand-initiated Nuclear Hormone Receptor Pharmacology, Receptor Selectivity, and Heterodimer Functionality

Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo- and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.

A Role for Rat Inositol Polyphosphate Kinases rIPK2 and rIPK1 in Inositol Pentakisphosphate and Inositol Hexakisphosphate Production in Rat-1 Cells

Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.