Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-1

Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E₂. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E₂ production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH₂, or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.     

Small Ubiquitin-Like Modifier Conjugating Enzyme with Active Site Mutation Acts as Dominant Negative Inhibitor of SUMO Conjugation in Arabidopsis

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.     

Interaction of protein kinase C with phosphatidylserine. 1. Cooperativity in lipid binding.

The basis for the apparent cooperativity in the activation of protein kinase C by phosphatidylserine has been addressed using proteolytic sensitivity, resonance energy transfer, and enzymatic activity. We show that binding of protein kinase C to detergent-lipid mixed micelles and model membranes is cooperatively regulated by phosphatidylserine. The sigmoidal dependence on phosphatidylserine for binding is indistinguishable from that observed for the activation of the kinase by this lipid [Newton & Koshland (1989) J. Biol. Chem. 264, 14909-14915]. Thus, protein kinase C activity is linearly related to the amount of phosphatidylserine bound. Furthermore, under conditions where protein kinase C is bound to micelles at all lipid concentrations, activation of the enzyme continues to display a sigmoidal dependence on the phosphatidylserine content of the micelle. This indicates that the apparent cooperativity in binding does not arise because protein kinase C senses a higher concentration of phosphatidylserine once recruited to the micelle. Our results reveal that the affinity of protein kinase C for phosphatidylserine increases as more of this lipid binds, supporting the hypothesis that a domain of phosphatidylserine is cooperatively sequestered around the enzyme.     

Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH₂-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K_i value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn³²–Met³⁸ and Gly⁹⁷–Leu¹⁰³, in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val³⁶-centerd hydrophobic cluster within the Asn³²–Met³⁸ region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile³⁵-Val³⁶-Tyr³⁷, in the Asn³²–Met³⁸ region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.     

High-Density Lipoprotein Inhibits UDP-N-Acetylgalactosamine:GM3,N-Acetylgalactosaminyltransferase and Differentiation of Cultured Cerebral Cells: Comparison with a Formerly Described Inhibitor of This Enzyme

Abstract: We have previously described a thermostable inhibitor of the UDP-N-acetylgalactosamine:GM3,N-acetylgalactosaminyltransferase (GM2 synthase) purified from chicken blood serum. Some properties of the GM2 synthase inhibitory preparation (IP) resemble those of high-density lipoprotein (HDL), i.e., both have a MW of 200,000 in native conditions and are resistant to denaturation by heat. These and other facts prompted us to test the possibility that lipoproteins regulate ganglioside biosynthesis in the CNS. For this purpose, serum lipoprotein fractions were isolated from chicken serum by flotation and were assayed as inhibitors of GM2 synthase activity and of neuron differentiation in culture. HDL (in contrast to fractions containing very low-density or low-density lipoprotein) inhibited GM2 synthase with the same specific activity as IP and inhibited neuron cell differentiation in culture in a similar way. Furthermore, these two preparations also share several other characteristics; i.e., both have the same cholesterol content, the same floating behavior on KBr gradients, and the same polypeptide pattern as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie Blue, or after western blot and revealing with an antibody prepared against IP, which is able to diminish the inhibitory effect of this preparation. The results described indicate identity between HDL and IP and suggest that HDL (particularly apolipoprotein A) could play an important role on ganglioside biosynthesis modulation during CNS development. The antineuritogenic effect of HDL described in this study could be of physiological relevance during CNS development and response to injury.     

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys¹³) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.     

Interaction of histone H1 molecules in a solution

Molecules of histones H1 isolated from the calf thymus, carp testicles and spermatozoa as well as trypsin-stable fragments of these proteins have been studied from the standpoint of their structure and interaction using methods of differential spectrophotometry, gel filtration and turbidimetry. The globular structure of histone H1 of the calf thymus is formed with an increase in the ionic strength of the medium and it is eluted as dimer with gel chromatography. With a considerable local increase of ionic strength (by addition of NaCl crystals) molecules of histones H1 form high-molecular aggregates from all the studied tissues. This aggregation is a result of interaction of globular trypsin-stable sites. Molecules of histone H1 from carp testicles and spermatozoa as well as their trypsin-stable fragments revealed no differences in the ability to form dimers and aggregates.     

Tumor Volume Can Be Used as a Parameter Indicating the Severity of Disease in Parathyroid Cancer

ObjectiveTo determine whether tumor volume and tumor size are related to disease severity in parathyroid cancer (PC).MethodsPatients treated for PC at our institution were retrospectively identified. Data were collected about clinical and pathologic characteristics, laboratory parameters, tumor volume, recurrence, metastasis, and mortality. Correlation analysis was applied to laboratory parameters, tumor volume, and tumor size in PC patients.ResultsThe study included 20 patients diagnosed with PC at our center. The median follow-up was 33 months. Serum calcium (median, 12.5 mg/dL), serum parathormone (PTH) (median, 743 pg/mL), and serum alkaline phosphatase (ALP) (median, 298 U/L) levels were found to be increased, and 25-hydroxyvitamin D (25[0H)D) (median, 12.3 ng/mL) and serum phosphorus (median, 2.1 mg/dL) levels were decreased. Magnesium level was within normal limits (median, 1.9 mg/dL). The median tumor volume was 5.7 mL and median tumor size was 2.5 cm. Significant positive correlations were found between tumor volume and calcium, ALP, and PTH levels. A significant negative correlation was found between tumor volume and 25(OH)D level. There were no significant correlations between tumor size and calcium, ALP, PTH, and 25(OH)D.ConclusionThese results found that the tumor volume affected PTH, calcium, ALP, and 25(OH)D levels. The morbidity and mortality associated with PC were usually associated with PTH secretion and hypercalcemia. Therefore, tumor volume may be a more effective parameter than tumor size when evaluating the severity of disease.     

MDA5 Can Be Exploited as Efficacious Genetic Adjuvant for DNA Vaccination against Lethal H5N1 Influenza Virus Infection in Chickens

Chickens lack the retinoic acid-inducible gene I (RIG-I) and sense avian influenza virus (AIV) infections by means of the melanoma differentiation-associated gene 5 product (chMDA5). Plasmid-driven expression of the N-terminal half of chMDA5 containing the caspase activation and recruitment domains [chMDA5(1-483)] triggers interferon-β responses in chicken cells. We hypothesized that mimicking virus infection by chMDA5(1-483) expression may enhance vaccine-induced adaptive immunity. In order to test this, the potential genetic adjuvant properties of chMDA5(1-483) were evaluated in vivo in combination with a suboptimal quantity of a plasmid DNA vaccine expressing haemagglutinin (HA) of H5N1 AIV. Co-administration of the HA plasmid with plasmid DNA for chMDA5(1-483) expression resulted in approximately 10-fold higher HA-specific antibody responses than injection of the HA plasmid mixed with empty vector DNA as control. Accordingly, compared with HA DNA vaccination alone, the chMDA5(1-483)-adjuvanted HA DNA vaccine mediated enhanced protection against a lethal H5N1 challenge infection in chickens, with reduced clinical signs and cloacal virus shedding. These data demonstrate that innate immune activation by expression of signaling domains of RIG-I-like receptors can be exploited to enhance vaccine efficacy.     

Genetic Mapping Reveals that Sinefungin Resistance in Toxoplasma gondii Is Controlled by a Putative Amino Acid Transporter Locus That Can Be Used as a Negative Selectable Marker

Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDR^r and type 10 VAND-SNF^r. The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies in T. gondii.     

ADP Is a Competitive Inhibitor of ATP-Dependent H Transport in Microsomal Membranes from Zea mays L. Coleoptiles

An anion-sensitive ATP-dependent H⁺ transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of the H⁺ transport observed: strict dependence on Cl⁻; inhibition by SO₄²⁻ and NO₃⁻; insensitivity against vanadate, molybdate, and azide; reversible inhibition by CaCl₂ (H⁺/Ca²⁺ antiport); inhibition by diethylstilbestrol. The substrate kinetics revealed simple Michaelis Menten kinetics for ATP in the presence of 1 millimolar MgCl₂ with a K_m value of 0.56 millimolar (0.38 millimolar for MgATP). AMP and c-AMP did not influence H⁺ transport significantly. However, ADP was a potent competitive inhibitor with a K_i value of 0.18 millimolar. The same inhibition type was found for membranes prepared from primary roots by the same procedure.     

Identification of the Matriptase Second CUB Domain as the Secondary Site for Interaction with Hepatocyte Growth Factor Activator Inhibitor Type-1

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1–58K) revealed that the second CUB domain (CUB domain II, Cys³⁴⁰–Pro⁴⁵²) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys³⁷⁹ and Val³⁸⁰ within CUB domain II and that the C-terminal residues after Val³⁸⁰ are responsible for facilitating the interaction with HAI-1–58K. A synthetic peptide corresponding to Val³⁸⁰–Asp³⁹⁰ markedly increased the matriptase-inhibiting activity of HAI-1–58K, whereas the peptides corresponding to Val³⁸⁰–Val³⁸⁹ and Phe³⁸²–Asp³⁹⁰ had no effect. HAI-1–58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val³⁸⁰–Pro³⁹² with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.     

A Dominant Negative Mutant of Pma1 Interferes with the Folding of the Wild Type Enzyme

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1 , the gene coding for the plasma membrane H ⁺-ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co-expressed with the wild type allele, both proteins are retained in the ER and degraded. We have used a PMA1 -D378T dominant lethal allele to analyse the mechanism underlying the retention of the wild type enzyme by the dominant negative mutant. A genetic screen was performed for isolation of intragenic suppressors of PMA1 -D378T allele. This analysis pointed to transmembrane helix 10 (TM10) as an important element in the establishment of the dominant lethality. Deletion of the TM10 was able to suppress not only the PMA1 -D378T but all the dominant lethal alleles tested. Biochemical analyses suggest that dominant lethal proteins obstruct, through TM10, the correct folding of the wild type enzyme leading to its retention and degradation by ERAD.     

Interaction of DNA with sperm-specific histones of the H1 family

Interactions of DNA with sperm-specific histones of the H1 family of sea urchin Strongylocentrotus intermedius, sea star Aphelasterias japonica, and bivalve mollusc Chlamis islandicus were studied using circular dichroism and the DNA melting analysis. Under physiological conditions, the highest DNA compacting ability was found in the echinoderm sperm H1 protein, in which additional α-helical domains are present in their C-terminal sequence. The derivative melting curves have two peaks: the low-temperature peak corresponds to the melting of free DNA, whereas the DNA regions bound to the protein melt at higher temperature. The highest stabilizing ability is characteristic of complexes with the mollusc sperm H1 protein.     

Differences in the binding of H1 variants to DNA. Cooperativity and linker-length related distribution

A study of the complexes formed between short linear DNA and three H1 variants, a typical somatic H1, and the extreme variants H5, from chicken erythrocytes, and spH1 from sea urchin sperm, has revealed differences between H1, H5 and spH1 that have implications for chromatin structure and folding. 1. All three histones bind cooperatively to DNA in 35 mM NaCl forming similar, but not identical, rod-like complexes. With sufficiently long DNA the complexes may be circular, circles forming more easily with H5 and spH1 than with H1. 2. The binding of H5 and spH1 to DNA is cooperative even in 5 mM NaCl, resulting in well-defined thin filaments that appear to contain two DNA molecules bridged by histone molecules. In contrast, H1 binds distributively over all the DNA molecules in 5 mM NaCl, but forms short stretches similar in appearance to the thin filaments formed with H5 and spH1. Rods appear to arise from the intertwining of regular thin filaments containing cooperatively bound histone molecules on raising the NaCl concentration to 35 mM. 3. The compositions of the rods correspond to one histone molecule for about every 47 bp (H1), 81 bp (H5) and 112 bp (spH1), suggesting average spacings of 24 bp (H1), 41 bp (H5) and 56 bp (spH1) in the component thin (double) filaments. Strikingly, these values are proportional to the linker lengths of the chromatins in which the particular H1 variant is the main or sole H1.     

Interaction of haemoglobin and cytochrome c with aliphatic alcohols as studied by 1H NMR spectroscopy

Association of haeme proteins, haemoglobin and cytochrome c, with eight aliphatic alcohols (methanol, ethanol. two isomeric propanols and four butanols) was studied by 1H NMR spectroscopy. NMR spectra of alcohols were monitored at 60 MHz at increasing concentration of the proteins. Selective broadening of the NMR signals of individual segments of alcohols was observed only in the case of alcohol-haemoglobin systems. Its quantitative evaluation and interpretation in terms of formation of low affinity intermolecular alcohol--protein complexes led to the conclusion that haemoglobin associates with alcohol molecules in a way depending on the length and isomeric branching of the alkyl chains; in particular, the methylene and methine groups vicinal to the hydroxyl are subject to stronger immobilization than the terminal methyls or other groups. Thus, the model of hydrophobic complexes stabilized by hydrogen bonds described previously for association of bovine serum albumin with alcohols (Lubas et al., Biochemistry, 18, 4943-4951, 1979) seems to apply also to haemoglobin association. In the case of cytochrome c association, 1H NMR data alone are insufficient for structural evaluation of the mechanism of formation of the alcohol--cytochrome c complexes.     

Evaluation of an Allosteric BACE Inhibitor Peptide to Identify Mimetics that Can Interact with the Loop F Region of the Enzyme and Prevent APP Cleavage

The aspartyl protease BACE1 (BACE) has emerged as an appealing target for reduction of amyloid-β in Alzheimer's disease. The clinical fate of active-site BACE inhibitors may depend on potential side effects related to enzyme and substrate selectivity. One strategy to reduce this risk is through development of allosteric inhibitors that interact with and modulate the Loop F region unique to BACE1. Previously, a BACE-inhibiting antibody (Ab) was shown by co-crystallization to bind and induce conformational changes of Loop F, resulting in backbone perturbations at the distal S6 and S7 subsites, preventing proper binding of a long APP-like substrate to BACE and inhibiting its cleavage. In an effort to discover small Loop F-interacting molecules that mimic the Ab inhibition, we evaluated a peptide series with a YPYF(I/L)P(L/Y) motif that was reported to bind a BACE exosite. Our studies show that the most potent inhibitor from this series, peptide 65007, has a similar substrate cleavage profile to the Ab and reduces sAPPβ levels in cell models and primary neurons. As our modeling indicates, it interacts with the Loop F region causing a conformational shift of the BACE protein backbone near the distal subsites. The peptide-bound enzyme adopts a conformation that closely overlays with the crystal structure (PDB: 3R1G) from Ab binding. Importantly, peptide 65007 appears to be BACE substrate and enzyme selective, showing little inhibition of NRG1, PSGL1, CHL1, or Cat D. Thus, peptide 65007 is a promising lead for discovery of Loop F-interacting small-molecule mimetics as allosteric inhibitors of BACE.     

RNA-Mediated Dimerization of the Human Deoxycytidine Deaminase APOBEC3H Influences Enzyme Activity and Interaction with Nucleic Acids

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4 + T cells. When aberrantly expressed in lung or breast tissue, APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12-nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, and deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single-molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA-mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.