ABSTRACT
Endospore-forming bacteria are ubiquitous. Bacterial endospores are multilayered proteinaceous structures that protects the bacterial genome during stress conditions. They are also responsible for a wide range of critical clinical infections in humans. Precise analysis of spore-forming pathogens remains a major challenge in the field of proteomics because spore structures are highly resistant to conventional solubilizers and denaturing agents, such as sodium dodecyl sulfate and urea. We present an ionic liquid-assisted (i-soln) technique of sample preparation, called pTRUST, which enables shotgun analysis of Bacillus subtilis spores even when the starting materials are in the sub-microgram range. In proteomic analysis, this technique shows 50-2000-fold higher sensitivity than other conventional gel-based or gel-free methods (including one-pot sample processing). Using this technique, we identified 445 proteins with high confidence from trace amounts of highly pure spore preparations, including 52 of the 79 proteins (approximately 70%) previously demonstrated to be localized in spores in the SubtiWiki database and detected through direct protein analysis. Consequently, 393 additional proteins were identified as candidates for spore constitutive proteins. Twenty of these newly identified candidates were produced as green fluorescent protein fusion proteins, and each was evaluated for authenticity as a spore constituent using fluorescence microscopy analysis. The pTRUST method's sensitivity and reliability using the i-soln system, together with hitherto unreported proteins in spores, will enable an array of spore research for biological and clinical applications.
Subject(s)
Bacillus subtilis , Bacterial Proteins , Ionic Liquids , Proteomics , Spores, Bacterial , Bacillus subtilis/metabolism , Spores, Bacterial/metabolism , Proteomics/methods , Bacterial Proteins/metabolism , Ionic Liquids/chemistry , Proteome/analysis , Proteome/metabolismABSTRACT
The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.
Subject(s)
Boronic Acids , Cryoelectron Microscopy , Proteasome Endopeptidase Complex , Proteasome Inhibitors , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/chemistry , Animals , Proteasome Inhibitors/pharmacology , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/chemical synthesis , Mice , Boronic Acids/chemistry , Boronic Acids/pharmacology , Boronic Acids/chemical synthesis , Humans , Structure-Activity Relationship , Cysteine Endopeptidases/metabolism , Molecular Structure , Molecular Dynamics Simulation , Drug DiscoveryABSTRACT
Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.
Subject(s)
Ionic Liquids , Proteome , Humans , Proteome/metabolism , Proteomics/methods , Chromatography, Liquid/methods , Membrane Proteins/metabolismABSTRACT
The renin-angiotensin-aldosterone system (RAAS) plays a key role in the regulation of blood pressure. Renin, the first and rate-limiting enzyme of the RAAS, is an attractive target for the treatment of hypertension and cardiovascular/renal diseases. Therefore, various direct renin inhibitors (DRIs) have been researched over recent decades; however, most exhibited poor pharmacokinetics and oral bioavailability due to the peptidomimetic or nonpeptidomimetic structures with a molecular weight (MW) of >600, and only aliskiren is approved. This study introduces a novel class of DRIs comprised of a 2-carbamoyl morpholine scaffold. These compounds have a nonpeptidomimetic structure and a MW of <500. The representative compound 26 was highly potent despite not occupying S1'-S2' sites or the opened flap region used by other DRIs and exerted a significant antihypertensive efficacy via oral administration on double transgenic mice carrying both the human angiotensinogen and the human renin genes.
ABSTRACT
Renin is the rate-limiting enzyme in the renin-angiotensin-aldosterone system (RAAS) which regulates blood pressure and renal function and hence is an attractive target for the treatment of hypertension and cardiovascular/renal diseases. However, the development of direct renin inhibitors (DRIs) with favorable oral bioavailability has been a longstanding challenge for many years. This problem was thought to be because most of the reported DRIs were peptide-like structures or nonpeptide-like structures with a molecular weight (MW) of > 600. Therefore, we tried to find nonpeptidomimetic DRIs with a MW of < 500 and discovered the promising 2-carbamoyl morpholine derivative 4. In our efforts to improve the pharmacokinetic profile of 4 without a significant increase in the MW, we discovered compound 18 (SPH3127), which demonstrated higher bioavailability and a more potent antihypertensive effect in preclinical models than aliskiren and has completed a phase II clinical trial for essential hypertension.
Subject(s)
Hypertension , Renin , Amides/pharmacology , Amides/therapeutic use , Antihypertensive Agents/pharmacology , Antihypertensive Agents/therapeutic use , Fumarates/pharmacology , Fumarates/therapeutic use , Humans , Hypertension/drug therapy , Morpholines/pharmacology , Renin/pharmacology , Renin/therapeutic use , Renin-Angiotensin SystemABSTRACT
Low-resolution electron density maps can pose a major obstacle in the determination and use of protein structures. Herein, we describe a novel method, called quality assessment based on an electron density map (QAEmap), which evaluates local protein structures determined by X-ray crystallography and could be applied to correct structural errors using low-resolution maps. QAEmap uses a three-dimensional deep convolutional neural network with electron density maps and their corresponding coordinates as input and predicts the correlation between the local structure and putative high-resolution experimental electron density map. This correlation could be used as a metric to modify the structure. Further, we propose that this method may be applied to evaluate ligand binding, which can be difficult to determine at low resolution.
Subject(s)
Proteins/chemistry , Crystallography, X-Ray/methods , Machine Learning , Neural Networks, ComputerABSTRACT
N1-Acetylspermine oxidase (APAO) catalyzes the conversion of N1-acetylspermine or N1-acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N1-acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.
Subject(s)
Oxidoreductases/chemistry , Putrescine/chemistry , Spermidine/analogs & derivatives , Spermidine/chemistry , Spermine/analogs & derivatives , Aldehydes/chemistry , Aldehydes/metabolism , Animals , Catalytic Domain , Gene Expression , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Kinetics , Mice , Models, Molecular , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Propylamines/chemistry , Propylamines/metabolism , Protein Structure, Secondary , Putrescine/analogs & derivatives , Putrescine/metabolism , Spermidine/metabolism , Spermine/chemistry , Spermine/metabolismABSTRACT
Optimization of a new series of S-adenosyl-L-homocysteine hydrolase (AdoHcyase) inhibitors based on non-adenosine analogs led to very potent compounds 14n, 18a, and 18b with IC50 values of 13 ± 3, 5.0 ± 2.0, and 8.5 ± 3.1 nM, respectively. An X-ray crystal structure of AdoHcyase with NAD(+) and 18a showed a novel open form co-crystal structure. 18a in the co-crystals formed intramolecular eight membered ring hydrogen bond formations. A single crystal X-ray structure of 14n also showed an intramolecular eight-membered ring hydrogen bond interaction.
Subject(s)
Adenosylhomocysteinase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Adenosine/chemistry , Adenosylhomocysteinase/genetics , Adenosylhomocysteinase/metabolism , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Hydrogen Bonding , Isomerism , Molecular Conformation , Molecular Dynamics Simulation , NAD/chemistry , NAD/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Structure-Activity RelationshipABSTRACT
BACKGROUND: There has been a growing need for a cognitive assessment tool that can be used for older adults with schizophrenia in clinical settings. The clock-drawing test (CDT) is a brief cognitive test that covers a wide range of cognitive function. Although it is widely used to assess patients with dementia, limited data are available on its usefulness in older patients with schizophrenia. Thus, we investigated the psychometric properties of the CDT and their relationship with life functions to examine the test's usefulness for assessing cognitive function in older adults with schizophrenia. METHODS: Seventy-three older adults with chronic schizophrenia who had been hospitalized for over 1 year participated in the study. We adopted the executive clock-drawing task for administration and scoring of the CDT, which consists of free-drawn and copy conditions. The Mini-Mental State Examination and the Brief Assessment of Cognition in Schizophrenia were administered. Symptom severity and life functions were assessed with the Positive and Negative Syndrome Scale and the Life Skills Profile, respectively. RESULTS: Both free-drawn and copy scores significantly correlated with the Mini-Mental State Examination score and the Brief Assessment of Cognition in Schizophrenia composite score. These scores also significantly correlated with symptom severity and length of current hospitalization. Stepwise regression analysis showed that only the copy score, together with symptom severity, predicted the Life Skills Profile score. CONCLUSIONS: The CDT can assess cognitive function in older adults with schizophrenia. Moreover, CDT performance is associated with life functions independent from other clinical variables. These results suggest that the CDT is a useful cognitive assessment tool for this population.
Subject(s)
Cognition Disorders/diagnosis , Geriatric Assessment , Institutionalization , Neuropsychological Tests/statistics & numerical data , Psychiatric Status Rating Scales/statistics & numerical data , Schizophrenia/diagnosis , Activities of Daily Living , Aged , Aged, 80 and over , Cognition Disorders/complications , Cognition Disorders/psychology , Female , Humans , Male , Mental Status Schedule , Middle Aged , Psychometrics , Psychomotor Performance , Regression Analysis , Reproducibility of Results , Schizophrenia/complications , Severity of Illness IndexABSTRACT
L-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6, which catalyzes the oxidative deamination of L-glutamate, has attracted increasing attention as a component of amperometric L-glutamate sensors used in the food industry and clinical biochemistry. The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an alpha(2)beta(2)V(2) structure; enzymatic proteolysis of the precursor forms the stable mature enzyme. We solved the crystal structure of mature LGOX using molecular replacement with a structurally homologous model of L-amino acid oxidase (LAAO) from snake venom: LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site. Comparison of the LGOX structure with that of LAAO revealed that LGOX has three regions that are absent from the LAAO structure, one of which is involved in the formation of the entrance. Furthermore, the arrangement of the residues composing the active site differs between LGOX and LAAO, and the active site of LGOX is narrower than that of LAAO. Results of the comparative analyses described herein raise the possibility that such a unique structure of LGOX is associated with its substrate specificity.
Subject(s)
Amino Acid Oxidoreductases/chemistry , Streptomyces/enzymology , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Molecular Structure , Molecular Weight , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Structural Homology, Protein , Substrate SpecificityABSTRACT
Human JNK stimulatory phosphatase-1 (JSP-1) is a novel member of dual specificity phosphatases. A C-terminus truncated JSP-1 was expressed in Escherichia coli and was crystallized using the sitting-drop vapor diffusion method. Thin-plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit-cell parameters a = 84.0 A, b = 49.3 A, c = 47.3 A, and beta = 119.5 degrees , and diffract up to 1.5 A resolution at 100 K. The structure of JSP-1 has a single compact (alpha/beta) domain, which consists of six alpha-helices and five beta-strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP-loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP-1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP-1 has no loop corresponding to the Lys120-loop of PTP1B, and tryptophan residue corresponding to the substrate-stacking in PTP1B is substituted by alanine residue in JSP-1.
Subject(s)
Phosphoprotein Phosphatases/chemistry , Protein Tyrosine Phosphatases/chemistry , Alkanesulfonic Acids/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Catalysis , Crystallography, X-Ray , Dual-Specificity Phosphatases , Humans , Mice , Mitogen-Activated Protein Kinase Phosphatases , Models, Molecular , Molecular Sequence Data , Morpholines/metabolism , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/isolation & purification , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Binding , Protein Conformation , Protein Phosphatase 1 , Protein Processing, Post-Translational , Protein Structure, Secondary , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/isolation & purification , Protein Tyrosine Phosphatases/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The Body Attitude Test (BAT) was developed by Probst et al. (1995) for female patients with eating disorders (ED). This test measures the subjective body experience and attitudes toward one's body. The present authors have developed the Japanese version of the BAT and the purpose of the present paper was to investigate its reliability and validity in control (CON, n = 599) and ED patients (n = 46). The ED patients consisted of 21 anorexia nervosa, restricting type (AN-R) patients and 25 bulimia nervosa (BN) patients. Internal consistency was determined with Cronbach's alpha coefficient in CON. Factor analysis was conducted on BAT ratings given by CON. Factor analysis indicated that BAT was composed of two factors. These were body dissatisfaction (factor 1) and lack of familiarity with one's body (factor 2). A comparison was made among AN-R, BN, and CON. Bulimia nervosa had a significantly higher score than the other two groups. The BAT scores of ED patients correlated significantly with the Self -rating Depression Scale, and State-Trait Anxiety Inventory. These results show that ED patients have negative feelings toward their own body, similar to the findings in the original report. On factor analysis, however, it was not possible to distinguish between negative appreciation of body size and general body dissatisfaction as described in the original report. The authors also examine influences on this difference from a cross-cultural view point.
Subject(s)
Body Image , Feeding and Eating Disorders/diagnosis , Language , Personality Inventory/statistics & numerical data , Adolescent , Adult , Female , Humans , Japan , Reproducibility of ResultsSubject(s)
Depression , Anti-Anxiety Agents/therapeutic use , Antipsychotic Agents/therapeutic use , Biogenic Monoamines , Depression/classification , Depression/diagnosis , Depression/etiology , Depression/therapy , Diagnosis, Differential , Diagnostic and Statistical Manual of Mental Disorders , Genetic Predisposition to Disease , Humans , Psychotherapy , Reference StandardsABSTRACT
We have introduced behavior therapy as standard in-patient treatment for anorexia nervosa and have modified the treatment program. At first, we used Fukamachi's activity restriction therapy (FT), followed by Token economy therapy (TET), which combined token economy with FT. Finally, we have developed Kyoto Prefectural University of Medicine Behavior Therapy (KPT). According to KPT, only liquid formula is given in the early stages of hospitalization and a target weight is not set at admission. We examined the effect of these three programs with respect to bodyweight gain. Thirty-five anorexic patients participated in these three programs in our hospital: seven completed FT, seven completed TET and 21 completed KPT. We compared the effects of these three programs on body mass index (BMI). Furthermore, the effects of these three programs on BMI were compared at admission, 1 month after admission and at discharge, 6 months after discharge. In addition, the rate of increase of BMI for the following three periods was investigated: 1 month after admission, total hospitalization (from admission to discharge) and from admission to 6 months after discharge. The result is that KPT was the most effective of the three programs with regard to both the amount and the rate of increase of BMI at all points and there is a significant difference between KPT and FT. This effectiveness may be attributable to the use of an oral liquid formula, the setting of target weight at a later stage of hospitalization and the release of activity restriction based on weight gain.