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Immobilizing DNA with high accessibility at the interface is attractive but challenging. Current methods often involve multiple chemical reactions and derivatives. In this study, an endonuclease, TC1, is introduced to develop a robust strategy for immobilizing DNA with enhanced accessibility. TC1 enables direct immobilization of DNA onto a solid support through self‐catalytic DNA covalent coupling and robust solid adsorption capabilities. This method demonstrates high accessibility to target molecules, supported by the improved sensitivity of DNA hybridization and aptamer‐target recognition assays. TC1‐mediated DNA immobilization is a one‐pot reaction that does not require chemical derivatives, making it promising for the development of high‐performance DNA materials and technologies. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) ultimately cause major adverse cardiovascular events (MACEs). While traditional strategies by lipid-reducing have reduced MACEs, many patients continue to face significant risks. It might attribute to the upregulation of CD47 expression in AS lesions, that mediated anti-efferocytosis of macrophages. Therefore, we propose simultaneously blocking ANGPTL3, a vital regulator of lipid metabolism, and CD47 might be a potential approach for AS therapy. Firstly, we investigate the role of a novel anti-ANGPTL3 nanobody-Fc (FD03) in AS. We found that FD03 treatment significantly decreased circulating lipids, plaque size, and lipid deposition in apoE-/- mice compared to control Ab, but there was a twofold increase in plaque formation in comparison to baseline. However, immunofluorescence indicated the upregulation of CD47 expression in the plaques even after FD03 treatment compared to normal vascular tissue. Next, a bifunctional protein containing signal regulatory protein alpha (SIRPα) and FD03 (SIRPαD1-FD03) was constructed to block CD47 and ANGPTL3 concurrently, which had high purity, robust stability, and high affinity to CD47 and ANGPTL3 with biological activity in vitro. Furthermore, SIRPαD1-FD03 fusion protein exhibited the enhanced therapeutic effect on AS compared with SIRPαD1-Fc or FD03, regressing plaque contents and the necrotic core equal to baseline. Mechanistically, SIRPαD1-FD03 reduced serum lipids, augmented the efferocytosis rate and macrophage M2 polarization, and decreased the reactive oxygen species (ROS) and lipid peroxidation level in atherosclerotic plaques. Collectively, our project suggests an effective approach for AS by simultaneously blocking ANGPTL3 and CD47 to regulate lipid metabolism, macrophage activity and lipid peroxidation.

Abstract Background Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. Methods Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsR M , was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25–6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. Results The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. Conclusions We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

Background: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. Methods: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25–6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. Results: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. Conclusions: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster. [ABSTRACT FROM AUTHOR]

Cell wall-anchored LPxTG proteins derived from the Lactobacillus are recognized to have excellent adhesion and gastrointestinal tolerance functions. Since the motif of LPxTG can interact with sortase A (SrtA) on the surface of Lactobacillus , it has great potential for various biotechnological applications. In this study, a bifunctional protein named LPxT-GYLEQ was designed by replacing the C-terminal hydrophobic domain with a synthetic antioxidant peptide containing an N-terminal glycine. It was found that the synthetic LPxT-GYLEQ protein exhibited better antioxidant activity in the H 2 O 2 -damaged HepG2 cells model. Meanwhile, it found that LPxT-GYLEQ protein can also exhibit good gastrointestinal tolerance function when pretreated with gastric juice and intestinal juice, and the DPPH radical scavenging ability of the LPxT-GYLEQ protein was increased by 12.8 % and 13.7 %, respectively, compared to the synthetic GYLEQ peptide. Further results also proved the good adhesion property of this synthetic protein (50 μg/mL) in the intestinal cell model. The novel strategy design of the antioxidative and adhesive bifunctional protein with the LPxTG motif structure offers a new idea for the protection of antioxidant peptides in the host oral delivery system. [Display omitted] • A bifunctional protein with antioxidant and intestinal adhesion properties was developed. • LPxT-GYLEQ protein possesses the ability of gastrointestinal tolerance and protection against antioxidant activity. • LPxTG-motif structure provides a new idea for the protection of functional peptides in the oral delivery system. [ABSTRACT FROM AUTHOR]

Biotin is an essential coenzyme involved in various metabolic processes across all known organisms, with biotinylation being crucial for the activity of carboxylases. BirA from Haemophilus influenzae is a bifunctional protein that acts as a biotin protein ligase and a transcriptional repressor. This study reveals the crystal structures of Hin BirA in both its apo- and holo-(biotinyl-5′-AMP bound) forms. As a class II BirA, it consists of three domains: N-terminal DNA binding domain, central catalytic domain, and C-terminal SH3-like domain. The structural analysis shows that the biotin-binding loop forms an ordered structure upon biotinyl-5′-AMP binding. This facilitates its interaction with the ligand and promotes protein dimerization. Comparative studies with other BirA homologs from different organisms indicate that the residues responsible for binding biotinyl-5′-AMP are highly conserved. This study also utilized AlphaFold2 to model the potential heterodimeric interaction between Hin BirA and biotin carboxyl carrier protein, thereby providing insights into the structural basis for biotinylation. These findings enhance our understanding of the structural and functional characteristics of Hin BirA, highlighting its potential as a target for novel antibiotics that disrupt the bacterial biotin synthesis pathways. • Our study characterized the crystal structure of BirA from Haemophilus influenzae. • Hin BirA is a bifunctional protein that acts as a biotin protein ligase (BPL) and a transcriptional repressor. • The ligand binding site and heterodimeric model of Hin BirA were analyzed. [ABSTRACT FROM AUTHOR]

Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and pyrophosphatase activities. Here we report the crystal structure of full-length hFADS2 and its C-terminal PAPS domain in complex with flavin adenine dinucleotide (FAD), and dissect the structural determinants underlying the contribution of each individual domain, within isoforms 1 and 2, to each of the two enzymatic activities. Structural and functional characterization performed on complete or truncated constructs confirmed that the C-terminal domain tightly binds FAD and catalyzes its synthesis, while the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. hFADS2 associates in a stable C2-symmetric dimer, in which the packing of the KH domain of one protomer against the N-terminal domain of the other creates the adenosine-specific active site responsible for the hydrolytic activity. [Display omitted] • Crystal structures of the bifunctional enzyme hFADS2 were determined • hFADS2 forms a stable C2-symmetric dimer involving the N-ter MPTb and KH domains • The N-terminal MPTb and KH domains are essential for FAD hydrolysis Leo et al. report the crystal structures of the full-length protein and the C-terminal domain of human FADS2. Functional characterization revealed that hFADS2 is a bifunctional enzyme responsible for both FAD formation and degradation that are catalyzed by different domains. The degradation activity requires the dimeric form of the protein. [ABSTRACT FROM AUTHOR]

Reduced brain levels of docosahexaenoic acid (C22:6n-3), a neurotrophic and neuroprotective fatty acid, may contribute to cognitive decline in Alzheimer's disease. Here, we investigated whether the liver enzyme system that provides docosahexaenoic acid to the brain is dysfunctional in this disease. Docosahexaenoic acid levels were reduced in temporal cortex, mid-frontal cortex and cerebellum of subjects with Alzheimer's disease, compared to control subjects (P = 0.007). Mini Mental State Examination (MMSE) scores positively correlated with docosahexaenoic/α-linolenic ratios in temporal cortex (P = 0.005) and mid-frontal cortex (P = 0.018), but not cerebellum. Similarly, liver docosahexaenoic acid content was lower in Alzheimer's disease patients than control subjects (P = 0.011). Liver docosahexaenoic/α-linolenic ratios correlated positively with MMSE scores (r = 0.78; P

Aims: Purification of porcine circovirus type 2 (PCV2) using Gram‐positive enhancer matrix (GEM) surface display technology and immunogenicity evaluation of the purified antigen. Methods and Results: A recombinant bifunctional protein containing a protein anchor domain and a 'virus anchor' domain was designed as a protein linker (PL) between PCV2 and GEM particles. By incubating with PL and GEM particles sequentially, PCV2 could be purified and enriched through a simple centrifugation process with GEM surface display technology. Our data showed that one unit (2·5 × 109 particles) of GEM particles with 80 μg PL could purify 100 ml of PCV2‐containing culture supernatant (viral titre: 106·5 TCID50 per ml−1) with a recovery rate up to 99·6%. The impurity removal efficiency of this method, calculated according to decreased total protein content during purification, was approximately 98%. Furthermore, in vivo experimentation showed that piglets immunized with purified PCV2 could elicit strong immune responses to prevent against PCV2 infection. Conclusion: Porcine circovirus type 2 could be efficiently purified and enriched with GEM display technology via a crucial PL, and the purified PCV2 could elicit effective immune responses against PCV2 infection. Significance and Impact of the Study: The GEM‐based purification method established here is cost‐efficient and high‐throughput, and may represent a promising large‐scale purification method for PCV2 vaccine production. [ABSTRACT FROM AUTHOR]

A new antibody-enzyme fusion protein, named Zipbodyzyme, composed of a Fab antibody (i.e., an antigen-binding fragment of an antibody) and an enzyme, has been successfully produced in the cytoplasm of Escherichia coli . Zipbodyzymes have a leucine zipper (LZ) pair at the C-termini of the heavy chain (Hc) and the light chain (Lc) of Fab, to promote the association of the Hc and the Lc in E. coli cytoplasm, adjoining a fused enzyme. A Zipbodyzyme containing mouse-derived anti- E. coli O157 Fab and a luciferase or a green fluorescent protein retained both the antigen-binding and an enzymatic activity/fluorescence. The bifunctional proteins were applicable in direct enzyme-linked immunosorbent assay (ELISA) without the need for a secondary antibody, indicating that the utility of the antibody enzyme bifunctional fusion protein will be expanded. [ABSTRACT FROM AUTHOR]

‘Candidatus Liberibacter solanacearum’ is an unculturable α-proteobacterium that is the putative causal agent of Zebra Chip (ZC) disease of potato. This disease is a major problem in potato growing areas in the United States and New Zealand, as it affects growth and yield of the crop which has resulted in millions of dollars of loss to the potato industries. ZC disease is characterised by browning and necrotic flecking of vascular and internal tissue, which when fried present as dark stripes and streaks within the chip rendering them commercially unacceptable. The potato-infecting clades of this bacterium are vectored by Bactericera cockerelli, the tomato potato psyllid. Vertical transmission via seed potatoes is another mechanism that can spread the disease. Current disease management strategies target the psyllid: as the pathogen is transmitted relativity quickly, these strategies are limited in control of the disease. Thus, new management strategies that target the bacterial pathogen are required. A number of high quality bacterial genomes are now available and comparative genomics has identified a number of potential targets. This bacterium has a relatively small, AT-rich genome that contains all the components of a type I secretion system, ABC transporters, as well as ten bifunctional protein genes that encode proteins with two different enzymatic domains. Two of the bifunctional genes encode proteins similar to those described as pathogenicity or virulence determinants in other organisms. The relevance of these bifunctional genes to pathogenicity and virulence of this species is discussed in relation to maintaining these domains in a relatively small, AT-rich genome and their putative pathogenicity/virulence roles. [ABSTRACT FROM AUTHOR]

Regulation of biological functions requires factors (proteins, peptides or chemicals) able to sense and translate environmental conditions or any circumstances in order to modulate the transcription of a gene, the stability of a transcript or the activity of a protein. Quorum sensing is a regulation mechanism connecting cell density to the physiological state of a single cell. In bacteria, quorum sensing coordinates virulence, cell fate and commitment to sporulation and other adaptation properties. The critical role of such regulatory systems was demonstrated in pathogenicity and adaptation of bacteria from the Bacillus cereus group (i.e. B. cereus and Bacillus thuringiensis). Furthermore, using insects as a model of infection, it was shown that sequential activation of several quorum sensing systems allowed bacteria to switch from a virulence state to a necrotrophic lifestyle, allowing their survival in the host cadaver, and ultimately to the commitment into sporulation. The chronological development of these physiological states is directed by quorum sensors forming the RNPP family. Among them, NprR combines two distinct functions connecting sporulation to necrotrophism in B. thuringiensis. In the absence of its cognate signaling peptide (NprX), NprR negatively controls sporulation by acting as a phosphatase. In the presence of NprX, it acts as a transcription factor regulating a set of genes involved in the survival of the bacteria in the insect cadaver.

Monoclonal antibodies enhance innate immunity, while bispecific T cell engager antibodies redirect adaptive T cell immunity. To stimulate both innate and adaptive mechanisms, we created a bifunctional eCD16A/anti-CD3-BFP adapter protein for combined use with clinically approved monoclonal IgG1 antibodies. The adaptor protein contains the extracellular domain of the human CD16A high-affinity variant, which binds the Fc domain of IgG1 antibodies, and an anti-human CD3 single-chain variable fragment that redirects T cell cytotoxicity. Functional characterization of eCD16A/anti-CD3-BFP was performed using in vitro, ex vivo, and in vivo animal assays. Combination treatments of eCD16A/anti-CD3-BFP with rituximab (anti-CD20), cetuximab (anti-EGFR), trastuzumab (anti-HER2) or anti-PD-L1 IgG1 led to specific killing of antigen-expressing tumor cells. In an ex vivo assay, eCD16A/anti-CD3-BFP combined with rituximab effectively eliminated B cells within peripheral blood samples from healthy donors and cancer patients; the effectiveness of this treatment was further enhanced by addition of either autologous or allogeneic γ9δ2 T cells. In mouse xenograft studies, the combination of eCD16A/anti-CD3-BFP and rituximab led to rapid depletion of tumor cells. The presence of non-targeting competing serum immunoglobulins interfered with efficacy, but this interference could be overcome by eCD16A/anti-CD3-BFP dose escalation or by using a Fc-glycoengineered version of rituximab. Moreover, combining eCD16A/anti-CD3-BFP with anti-Epstein Barr virus (EBV) antibodies directed T cell cytotoxicity toward EBV-infected cells. These findings support further development of eCD16A/anti-CD3-BFP as a novel anti-cancer therapeutic to stimulate both innate and adaptive immunity.

Many bacteria produce cytolytic toxins that target host cells or other competing microbes. It is well known that environmental factors control toxin expression, however recent work suggests that some bacteria manipulate the fold of these protein toxins to control their function. The β-sheet rich amyloid fold is a highly stable ordered aggregate that many toxins form in response to specific environmental conditions. When in the amyloid state, toxins become inert, losing the cytolytic activity they display in the soluble form. Emerging evidence suggest that some amyloids function as toxin storage systems until they are again needed, while other bacteria utilize amyloids as a structural matrix component of biofilms. This amyloid matrix component facilitates resistance to biofilm disruptive challenges. The bacterial amyloids discussed in this review reveal an elegant system where changes in protein fold and solubility dictate the function of proteins in response to the environment.

Primordial proteins, the evolutionary ancestors of modern sequences, are presumed to have been minimally active and nonspecific. Following eons of selective pressure, these early progenitors evolved into highly active and specific proteins. While evolutionary trajectories from poorly active and multifunctional generalists toward highly active specialists likely occurred many times in evolutionary history, such pathways are difficult to reconstruct in natural systems, where primordial sequences are lost to time. To test the hypothesis that selection for enhanced activity leads to a loss of promiscuity, we evolved a de novo designed bifunctional protein. The parental protein, denoted Syn-IF, was chosen from a library of binary patterned 4-helix bundles. Syn-IF was shown previously to rescue two different auxotrophic strains of E. coli: Δ ilvA and Δ fes. These two strains contain deletions for proteins with very different biochemical functions; IlvA is involved in isoleucine biosynthesis, while Fes is involved in iron assimilation. In two separate experiments, Syn-IF, was evolved for faster rescue of either Δ ilvA or Δ fes. Following multiple rounds of mutagenesis, two new proteins were selected, each capable of rescuing the selected function significantly faster than the parental protein. In each case, the evolved protein also lost the ability to rescue the unselected function. In both evolutionary trajectories, the original bifunctional generalist was evolved into a monofunctional specialist with enhanced activity. [ABSTRACT FROM AUTHOR]

In plant cells, the interconversion of formyl- and methylene-tetrahydrofolates is catalyzed by a bifunctional protein possessing methenyltetrallydrofolate cydohydrolase (EC 3.5.4.9) and methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. The present work reports the isolation and sequencing of a cDNA that encodes this protein. Polydonal antibodies, raised against purified pea cytosolic dehydrogenase:cyclohydrolase, were used to screen a λgt 11 cDNA expression library, constructed from leaf extracts of this species. The screen identified a phage containing a cDNA insert with an open reading frame encoding a 294 amino acid protein (Mr 31,344). The deduced primary structure of this protein contained most of the conserved regions found in other dehydrogenase:cyclohydrolase proteins including the corresponding domains of the trifunctional C1-THF synthases of mammalian and yeast origins.

In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilution gas chromatography negative chemical ionization mass spectrometry (GC-NCI-MS) methods for quantification of peroxisomal β-oxidation intermediates of pristanic acid in human plasma: 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid. The carboxylic groups of the intermediates were converted into pentafluorobenzyl esters, whereas hydroxyl groups were acetylated and ketogroups were methoximized. Hereafter, the samples were subjected to clean-up by high performance liquid chromatography. Analyses were performed by selected monitoring of the carboxylate anions of the derivatives. Control values of all three metabolites were established (2,3-pristenic acid: 2–48 nm, 3-hydroxypristanic acid: 0.02–0.81 nm, 3-ketopristanic acid: 0.07–1.45 nm). A correlation between the concentrations of pristanic acid and its intermediates in plasma was found. The diagnostic value of the methods is illustrated by measurements of the intermediates in plasma from patients with peroxisomal disorders. It is shown that in generalized peroxisomal disorders, the absolute concentrations of 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid were comparable to those in the controls, whereas relative to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein deficiency, elevated levels of 2,3-pristenic acid and 3-hydroxypristanic acid were found. 3-Ketopristanic acid, although within the normal range, was relatively low when compared to the high pristanic acid levels in these patients.—Verhoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jansen, H. J. ten Brink, R. J. A. Wanders, and C. Jakobs. Analysis of pristanic acid β-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry. J. Lipid Res. 1999. 40: 260–266.

Moonlighting proteins serve one or more novel functions in addition to their canonical roles. Moonlighting functions arise when an adventitious interaction between a protein and a new partner improves fitness of the organism. Selective pressure for improvement in the new function can result in two alternative outcomes. The gene encoding the newly bifunctional protein may duplicate and diverge so as to encode two proteins, each of which serves only one function. Alternatively, genetic changes that minimize adaptive conflict between the two functions and/or improve control over the time and place at which each function is served can lead to a moonlighting protein. Importantly, genetic changes that enhance a moonlighting function can occur in the gene encoding the moonlighting protein itself, in a gene that affects the structure of its new partner or in a gene encoding a transcription factor that controls expression of either partner. The evolutionary history of each moonlighting protein is complex, depending on the stochastic occurrence of genetic changes such as gene duplication and point mutations, and the effects of those changes on fitness. Population effects, particularly loss of promising individuals due to random genetic drift, also play a role in the emergence of a moonlighting protein. The ultimate outcome is not necessarily the 'optimal' solution to the problem of serving two functions, but may be 'good enough' so that fitness becomes limited by some other function. [ABSTRACT FROM AUTHOR]

Many bacteria produce cytolytic toxins that target host cells or other competing microbes. It is well known that environmental factors control toxin expression, however, recent work suggests that some bacteria manipulate the fold of these protein toxins to control their function. The β-sheet rich amyloid fold is a highly stable ordered aggregate that many toxins form in response to specific environmental conditions. When in the amyloid state, toxins become inert, losing the cytolytic activity they display in the soluble form. Emerging evidence suggest that some amyloids function as toxin storage systems until they are again needed, while other bacteria utilize amyloids as a structural matrix component of biofilms. This amyloid matrix component facilitates resistance to biofilm disruptive challenges.The bacterial amyloids discussed in this review reveal an elegant system where changes in protein fold and solubility dictate the function of proteins in response to the environment. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor (TNF) and internleukin-1 (IL-1) are the most potent proinflammatory cytokines involving in autoimmune and inflammatory human diseases. Many anti-inflammatory agents have been exploited for anti-inflammation treatments by targeting cytokines including TNF and IL-1. Theoretically, simultaneously neutralizing or blocking two important inflammatory mediators may achieve a synergistic therapeutic effect. We have developed a recombinant fusion protein, TNFR2-Fc-IL-1ra (TFI), which consists of a TNF-neutralizing domain that specifically binds to TNF-α, an IL-1 receptor antagonist domain, and a dimerization Fc portion of human IgG1, for bifunctional inflammatory inhibitor. Recombinant DNA expressing the sequence of this fusion protein was expressed in CHO-S cells. The protein product was purified using a two-step purification protocol and the identity of the protein was confirmed by western blot analysis. The purified recombinant protein had a purity of about 98 % as determined by HPLC, and a molecular mass of 164.6 kDa as determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The results of cell binding inhibition indicate that TFI was able to strongly neutralize TNF activity and antagonize IL-1r activity, suggesting that TFI may be used as a bifunctional ligand with enhanced anti-inflammatory effect. The result obtained in this study may provide a platform for extending bifunctional anti-inflammatory drug development. [ABSTRACT FROM AUTHOR]

Abstract: T-protein is composed of chorismate mutase (AroQT) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQT with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the β2-strand in forming a Gβ1 homo-dimerization interface explains the relevance of the first-β-hairpin in stabilizing the dimeric TyrA protein. [Copyright &y& Elsevier]

The enzyme homocitrate synthase (HCS) catalyzes the first step in lysine biosynthesis, and early biochemical data placed it in the cytoplasm or mitochondria, where most amino acid synthesis occurs. It was therefore surprising when refined fractionation techniques and specific immunoreagents clearly demonstrated its localization to the nucleus. These observations raised the question of whether HCS had a function within the nucleus independent of lysine synthesis.We demonstrate that HCS encoded by LYS20 in yeast is linked to the key process of DNA damage repair through the essential MYST family histone acetyltransferase Esa1 and the H2A.Z histone variant. This discovery indicates that HCS has a role in addition to amino acid synthesis, and that it functions in nuclear activities involving chromatin regulation that are distinct from its previously established role in lysine biosynthesis. The chromatin-linked roles are dependent on nuclear localization of Lys20, but are independent of HCS catalytic activity. Thus, Lys20 appears to have evolved as a bifunctional protein that connects cellular metabolism with chromatin functions. [ABSTRACT FROM AUTHOR]

Organophosphate (OP) neurotoxins have contaminated the environment, contributed to millions of poisoning annually, and have been used as chemical weapons. Biomaterials incorporating the native activity of the OP hydrolase (OPH) enzyme are of interest for applications including OP sensing, environmental bioremediation and prophylactic decontamination. We have engineered and characterized four novel hydrogel-forming OPH variants by genetically fusing the OPH enzyme with a-helical leucine zipper domains (H), unstructured soluble linker domains (S) and polyhistidine purification tags. The appended H domains form physical cross-links between the enzymes and enable self-assembly of the enzymes into hydrogels. The addition of the H and S fusions significantly increased the expression levels of soluble protein. OPH constructs with biterminal H domains form hydrogels at lower protein weight percents and exhibit higher enzymatic activity than those variants modified with a single H domain fusion. Polyhistidine tags were not useful for purification but they were not benign, as the addition of the 6His tags increased the hydrogelforming abilities of the proteins with a concomitant reduction in both the kcat and KM values. Active enzymatic hydrogels could be made from concentrated unpurified crude protein lysates, significantly simplifying the processing and utilization of the biomaterials. And, a simple proteinaceous bioactive surface coating exhibiting OPH activity is demonstrated. The hydrogels were stable over long-term storage, as activity was retained after cold storage in buffer after 5 months. These new protein constructs further show the use of rational protein design to create novel, bifunctional, self-assembling units for the formation of catalytic biomaterials. [ABSTRACT FROM AUTHOR]

Peroxisomes are ubiquitous cell organelles, highly abundant in the rat renal cortex. The distribution of peroxisomes in the epithelial cells of the nephron was demonstrated using the cytochemical DAB method. The rat proximal contort tubules contained a small number of peroxisomes, this appearance being different from man. The peroxisomes had an irregular shape due to the presence of tubules in the peripheral matrix but were devoid of other crystaline structures. The rat kiney peroxisomes had a rich enzimatic content consisting of catalase (metabolism of hydrogen peroxide ), bifunctional protein, (the fatty acids beta oxidation), dihidroxi-acetonephosphate-acyltransferase and alkyl-dihidroxi-acetonephosphate- sinthase (the biosynthesis of plasmalogens). All these enzymes were identified using the immunoelectronmicroscopy technique and were localised mostly in the peripheral matrix or along the peroxisomal membrane. They confirme the important role of peroxisomes in cell metabolism. [ABSTRACT FROM AUTHOR]

Abstract: The fusion of protein domains is an important mechanism in molecular evolution and a valuable strategy for protein engineering. We are interested in creating fusion proteins containing both globular and structural domains so that the final chimeric protein can be utilized to create novel bioactive biomaterials. Interactions between fused domains can be desirable in some fusion protein applications, but in this case the optimal configuration will enable the bioactivity to be unaffected by the structural cross-linking. To explore this concept, we have created a fusion consisting of a thermostable aldo-keto reductase, two α-helical leucine zipper domains, and a randomly coiled domain. The resulting protein is bifunctional in that (1) it can self-assemble into a hydrogel material as the terminal leucine zipper domains form interprotein coiled-coil cross-links, and (2) it expresses alcohol dehydrogenase and aldo-keto reductase activity native to AdhD from Pyrococcus furiosus. The kinetic parameters of the enzyme are minimally affected by the addition of the helical appendages, and rheological studies demonstrate that a supramolecular assembly of the bifunctional protein building blocks forms a hydrogel. An active hydrogel is produced at temperatures up to 60 °C, and we demonstrate the functionality of the biomaterial by monitoring the oxidation and reduction of the native substrates by the gel. The design of chimeric fusion proteins with both globular and structural domains is an important advancement for the creation of bioactive biomaterials for biotechnology applications such as tissue engineering, bioelectrocatalysis, and biosensing and for the study of native assembled enzyme structures and clustered enzyme systems such as metabolons. [Copyright &y& Elsevier]

Abstract: Blue fluorescent protein of aequorin (BFP) is a complex of Ca2+-bound apoaequorin with coelenteramide and is a bifunctional protein, which shows blue fluorescence and the luminescence activity like a luciferase. To reconstitute synthetic BFP (syn-BFP) from apoaequorin and coelenteramide, we established new synthetic route of coelenteramide and prepared highly purified recombinant aequorin using the histidine-tagged secretion system in Escherichia coli cells. As a result, we succeeded in reconstituting syn-BFP quantitatively and the fluorescence and luminescence properties of syn-BFP were identical to that of BFP obtained from aequorin. [Copyright &y& Elsevier]

Sulfite reductase (SiR) is an important enzyme catalyzing the reduction of sulfite to sulfide during sulfur assimilation in plants. This enzyme is localized in plastids, including chloroplasts, and uses ferredoxin as an electron donor. Ferredoxin-dependent SiR has been found in isolated chloroplast nucleoids, but its localization in vivo or in intact plastids has not been examined. Here, we report the DNA-binding properties of SiRs from pea (PsSiR) and maize (ZmSiR) using an enzymatically active holoenzyme with prosthetic groups. PsSiR binds to both double-stranded and single-stranded DNA without significant sequence specificity. DNA binding did not affect the enzymatic activity of PsSiR, suggesting that ferredoxin and sulfite are accessible to SiR molecules within the nucleoids. Comparison of PsSiR and ZmSiR suggests that ZmSiR does indeed have DNA-binding activity, as was reported previously, but the DNA affinity and DNA-compacting ability are higher in PsSiR than in ZmSiR. The tight compaction of nucleoids by PsSiR led to severe repression of transcription activity in pea nucleoids. Indirect immunofluorescence microscopy showed that the majority of SiR molecules colocalized with nucleoids in pea chloroplasts, whereas no particular localization to nucleoids was detected in maize chloroplasts. These results suggest that SiR plays an essential role in compacting nucleoids in plastids, but that the extent of association of SiR with nucleoids varies among plant species. [ABSTRACT FROM AUTHOR]

In order to find new insect resistance genes, four homologous cDNAs, hta-a, hta-b, hta-c and hta-d with lengths of 775, 718, 784 and 752 bp, respectively (GenBank accession numbers AF477031-AF477034), were isolated from a tuber cDNA expression library of Helianthus tuberosus L. Sequence analysis revealed that all four cDNAs contain an open reading frame of 444 bp, coding a polypeptide of 147 amino acid residues, and that the sequences of the cDNAs are very similar to those of the mannose-binding agglutinin genes of the jacalin-related family. In hemagglutination reactions and hapten inhibition assays, affinity-purified HTA (Helianthus tuberosus agglutinin) from induced Escherichia coli BL21(DE3) expressing GST-HTA shows hemagglutination ability and a higher carbohydrate-binding ability for mannose than other tested sugars. Trypsin inhibitory activity was detected in the crude extracts of induced E. coli BL21(DE3) expressing HTA, and was further verified by trypsin inhibitory activity staining on native polyacrylamide gel. The mechanism of interaction between HTA and trypsin was studied by molecular modeling. We found that plenty of hydrogen bonds and electrostatic interactions can be formed between the supposed binding sites of HTA-b and the active site of trypsin, and that a stable HTA/trypsin complex can be formed. The results above imply that HTA might be a bifunctional protein with carbohydrate-binding activity and trypsin inhibitory activity. Moreover, Northern blotting analysis demonstrated that hta is predominantly expressed in tubers of H. tuberosus, very weakly expressed in stems, but not expressed at all in other tissues. Southern blotting analysis indicated that hta is encoded by a multi-gene family. The insect resistance traits have been described in another paper. (Managing editor: Li-Hui Zhao) [ABSTRACT FROM AUTHOR]

Abstract: 4a-Hydroxy-tetrahydrobiopterin dehydratase/DCoH is a bifunctional protein. In the cytoplasm it is an enzyme required for the regeneration of tetrahydrobiopterin, an essential cofactor for phenylalanine hydroxylase. In the nucleus it functions as a transcriptional coactivator by forming a 2:2 heterotetramer with the hepatic nuclear factor HNF1α (HNF1). Patients with a deficiency of dehydratase activity have elevated levels of phenylalanine, and accumulate 7-pterins due to degradation of its substrate 4a-hydroxy-tetrahydrobiopterin. Curiously, the hyperphenylalaninemia is transient, and no defects in the transcriptional coactivator function have been reported. Recently, a human isozyme, dehydratase/DCoHα, has been detected which shares 60% identity with dehydratase/DCoH. This investigation was undertaken to ascertain if dehydratase/DCoHα has the pre-requisite properties to compensate in individuals lacking an active form of DCoH. DCoHα demonstrated the ability to quantitatively alter HNF1-dependent DNA-binding in vitro whereas DCoH was ineffective in vitro. This characteristic, due to the presence of dimeric DCoHα, demonstrates that DCoHα does not require any additional mammalian regulation process to alter DNA binding and therefore, may be more effective than DCoH at low concentrations. The dehydratase activity of each isoform was measured by a direct spectrophotometric assay. K m and V max for DCoHα were both 2–3 times higher than for DCoH, thus leaving the catalytic efficiency (V max/K m) the same for both enzymes. In conclusion, the properties of dehydratase/DCoHα are consistent with the hypothesis that the activity of this isozyme could account for the relatively mild symptoms reported for patients with a defect in dehydratase/DCoH. [Copyright &y& Elsevier]

Human lutropin (hLH) and human chorionic gonadotropin (hCG) are structurally and functionally similar and play important roles in reproduction via a common gonadal receptor (LH-R). However, hormone specific hCG-β subunit contains 24 additional amino acid carboxyl terminal peptide (CTP), which produce specific antibodies to hCG-β with little cross-reaction with LH. A chimeric protein containing both hLH-R and hCG-β would provide a unique bifunctional antigen for immunocontraception. In this study is described the synthesis of a chimeric DNA construct of full-length of hLH-receptor and hCG-β and its expression in Sf9 cells to produce a bifunctional protein. Recombinant protein was recognized by antibodies to LH-R as well as anti-hCG-β in Western Blots, thus indicating the preservation of immunological epitopes for both hLH-R and hCG-β in the chimera. Specific ligand binding of recombinant hLH-R component was demonstrated by the displacement of bound labeled hCG at increasing concentrations of unlabeled hCG, indicating that, the presence of hCG-β component of the chimera did not interfere with the binding of hCG to LH-R. hCG-β was also present in the recombinant chimeric protein as shown by a specific hCG-β chemiluminescence assay. Treatment of transfected Sf9 cells with hCG induced dose-dependent increase in the stimulation of intracellular cAMP production, which showed that the ligand binding had functional activity. These results demonstrate that the chimeric DNA construct of hLHR-hCG-β expressed a bifunctional protein containing both hLH-R and hCG-β activities, which could provide a unique potential antigen for immunocontraception in vertebrates. [Copyright &y& Elsevier]

A tetrapeptide, RGDS, was inserted into proUK kringle domain G118-L119 by the construction of a mutant proUK-RGDS gene. The gene was expressed in the baculovirus expression system. Immunoaffinity chromatography was used to purify the chimera and protein with purity over 90% was achieved. The chimera was tested for its platelet membrane binding function and showed a calcium-dependent platelet binding activity. Amidolytic activity of the chimera was tested. The result indicated that specific amidolytic activity of plasmin activated chimera was 62 000 IU/mg, comparable to the previously reported 65 355 IU/mg of plasmin activated natural proUK[1]. Activation of plasminogen by the chimera after plasmin treatment followed Michieal-Menten kinetics, and the Km was 0.97 μmol/L, which was also comparable to 1.64 μmol/L of native urokinase. The chimera also showed intensive ability to inhibit platelet aggregation in vitro. These results indicate that this chimera might be useful as a bifunctional thrombolytic agent. [ABSTRACT FROM AUTHOR]

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3α, 7α, 12α-trihydroxy-5β-cholestan-26-oic acid and 3α, 7α-dihydroxy-5β-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3α, 7α, 12α-trihydroxy-5β-cholest-24-en-26-oyl-CoA and 3α, 7α-dihydroxy-5β-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography. [ABSTRACT FROM AUTHOR]

The second and third reactions of the peroxisomal β-oxidation spiral are thought to be catalyzed by enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (L-bifunctional protein). Recently, we found the presence of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-bifunctional protein) in mammalian peroxisomes. Therefore, we studied the physiological role of the D-bifunctional protein. The contents of the L- and D-bifunctional proteins were about 0.01 and 0.5 μg/mg protein, respectively, in cultured human skin fibroblasts. The activity of conversion of hexadecenoyl-CoA to 3-ketopalmitoyl-CoA by the D-bifunctional protein was estimated to be about 0.5 milliunit/mg of fibroblast protein. This value was about 100-fold that of the L-bifunctional protein in the fibroblasts. From comparison of the activities of the bifunctional proteins with the rate of palmitate oxidation and the activities of acyl-CoA oxidase and 3-ketoacyl-CoA thiolase, it is proposed that the D-bifunctional protein plays a major role in the peroxisomal oxidation of palmitate in the fibroblasts. The contents of both the L- and D-bifunctional proteins in liver were about 2.5 μg/mg protein. Therefore, it is suggested that the D-bifunctional protein also plays a significant role in human liver peroxisomal fatty acid oxidation. Actions of the bifunctional proteins on enoyl forms of other acyl-CoA derivatives were examined. The D-bifunctional protein but not the L-bifunctional protein reacted with 2-methylhexadecenoyl-CoA and 3α,7α,12α-trihydroxy-5β-cholest-24-enoyl-CoA. We propose that, among the reactions of the distinct group of carboxylates oxidized specifically in peroxisomes, oxidation of 2-methyl-branched fatty acids and side-chain shortening of cholesterol for bile acid formation are catalyzed by the D-bifunctional protein, but not the L-bifunctional protein. [ABSTRACT FROM AUTHOR]

When D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein was purified from human liver, two preparations were obtained. One contained a 77-kDa polypeptide as the main component and minor smaller polypeptides including a 46-kDa polypeptide, and this preparation showed both the dehydratase and dehydrogenase activities. The other preparation was a homodimer of the 46-kDa polypeptide and showed only the dehydratase activity. Further analysis indicated that the native enzyme is a homodimer of 77-kDa polypeptide, which was proteolytically modified during purification. The cDNA for the human 77-kDa polypeptide was cloned. The amino acid sequences of the peptides derived from the components of the enzyme preparations were located in the deduced amino acid sequence of the cDNA. The preparation containing the 77-kDa polypeptide was treated with a protease, and two monofunctional fragments were separated. The dehydrogenase and dehydratase fragments were located on the amino- and carboxyl-terminal sides, respectively, of the deduced amino acid sequence of the cDNA. The protein expressed by the cDNA with the entire coding region exhibited both the dehydratase and dehydrogenase activities, and that expressed by a truncated version covering the carboxyl-terminal side exhibited only the dehydratase activity. The cloned cDNA was identical to the human 17β-hydroxysteroid dehydrogenase IV cDNA. [ABSTRACT FROM AUTHOR]

We have previously purified two D-3-hydroxyacyl-CoA dehydratase preparations from human liver. One preparation contained a 77-kDa polypeptide and smaller polypeptides, and the other was a homodimer of a 46-kDa polypeptide. Three different purified rat peroxisomal D-3-hydroxyacyl-CoA dehydratase preparations have been reported. Therefore, rat enzyme was purified in this study to confirm the enzyme structure. Two preparations with similar molecular structures to the human enzyme preparations were obtained, and these were similar to each other in immunochemical and catalytic properties. It was suggested that the native enzyme was a homodimer of the 77-kDa polypeptide, and this enzyme was modified to a homodimer of the 46-kDa polypeptide, because conversion of the 77-kDa polypeptide to smaller polypeptides including the 46-kDa polypeptide was clearly observed during purification. Rat liver subcellular fractionation study indicates that this enzyme is located in peroxisomes. The enzyme preparation containing the 77-kDa polypeptide catalyzed the D-3-hydroxyacyl-CoA dehydrogenase reaction as well as the dehydratase reaction. Thus, it is proposed that this enzyme is D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. [ABSTRACT FROM AUTHOR]

Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome. [ABSTRACT FROM AUTHOR]