Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.     

Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.     

Crosstalk between the subunits of the homodimeric enzyme triosephosphate isomerase

Homodimeric triosephosphate isomerase (TIM) from Trypanosoma cruzi (TcTIM) and T. brucei (TbTIM) are markedly similar in amino acid sequence and three-dimensional structure. In their dimer interfaces, each monomer has a Cys15 that is surrounded by loop3 of the adjoining subunit. Perturbation of Cys15 by methylmethane thiosulfonate (MMTS) induces abolition of catalysis and structural changes. In the two TIMs, the structural arrangements of their Cys15 are almost identical. Nevertheless, the susceptibility of TcTIM to MMTS is nearly 100-fold higher than in TbTIM. To ascertain the extent to which the characteristics of the interface Cys depend on the dynamics of its own monomer or on those of the adjacent monomer, we studied MMTS action on mutants of TcTIM that had the interface residues of TbTIM, and hybrids that have only one interfacial Cys15 (C15ATcTIM-wild type TbTIM). We found that the solvent exposure of the interfacial Cys depends predominantly on the characteristics of the adjoining monomer. The maximal inhibition of activity induced by perturbation of the sole interface Cys in the C15ATcTIM-TbTIM hybrid is around 60%. Hybrids formed with C15ATcTIM monomers and catalytically inert TbTIM monomers (E168DTbTIM) were also studied. Their activity drops by nearly 50% when the only interfacial Cys is perturbed. These results in conjunction with those on C15ATcTIM-wild type TbTIM hybrid indicate that about half of the activity of each monomer depends on the integrity of each of the two Cys15-loop3 portions of the interface. This could be another reason of why TIM is an obligatory dimer.     

Susceptibility to proteolysis of triosephosphate isomerase from two pathogenic parasites: characterization of an enzyme with an intact and a nicked monomer

The susceptibility to subtilisin of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) was studied. Their amino sequence and 3D structure are markedly similar. In 36 h of incubation at a molar ratio of 4 TIM per subtilisin, TcTIM underwent extensive hydrolysis, loss of activity, and large structural alterations. Under the same conditions, only about 50% of the monomers of TbTIM were cleaved in two sites. The higher sensitivity of TcTIM to subtilisin is probably due to a higher intrinsic flexibility. We isolated and characterized TbTIM that had been exposed to subtilisin. It exhibited the molecular mass of the dimer, albeit it was formed by one intact and one nicked monomer. Its k(cat) with glyceraldehyde 3-phosphate was half that of native TbTIM, with no change in K(m). The intrinsic fluorescence of nicked TbTIM was red-shifted by 5 nm. The association between subunits was not affected. The TbTIM data suggest that there are structural differences in the two monomers or that alterations of one subunit change the characteristics of the other subunit. In comparison to the action of subtilisin on TIMs from other species, the trypanosomal enzymes appear to be unique.     

Optimization of a β‐sheet‐cap for long loop closure

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: R W ITVTI – loop – KKIRV W E). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: R W ITVTI‐(GGGGKK)₂GGGG‐KKIRV W E) remaining folded (ΔG_U = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: R W ITVRI W ‐(GGGGKK)₂GGGG‐ W KTIRV W E) supports a remarkably stable β‐sheet (ΔG_U = 6.3 kJ/mol at 295 K, T_m = ～55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.     

β-turn tendency in N-methylated peptides with dehydrophenylalanine residue: DFT study

The tendency to adopt β‐turn conformation by model dipeptides with α,β‐dehydrophenylalanine (ΔPhe) residue in the gas phase and in solution is investigated by theoretical methods. We pay special attention to a dependence of conformational properties on the side‐chain configuration of dehydro residue and the influence of N‐methylation on β‐turn stability. An extensive computational study of the conformational preferences of Z and E isomers of dipeptides Ac‐Gly‐(E/Z)‐ΔPhe‐NHMe ( 1a / 1b ) and Ac‐Gly‐(E/Z)‐ΔPhe‐NMe₂ ( 2a / 2b ) by B3LYP/6‐311++G(d,p) and MP2/6‐311++G(d,p) methods is reported. It is shown that, in agreement with experimental data, Ac‐Gly‐(Z)‐ΔPhe‐NHMe has a great tendency to adopt β‐turn conformation. In the gas phase the type II β‐turn is preferred, whereas in the polar environment, the type I. On the other hand, dehydro residue in Ac‐Gly‐(E)‐ΔPhe‐NHMe has a preference to adopt extended conformations in all environments. N‐methylation of C‐terminal amide group, which prevents the formation of 1←4 intramolecular hydrogen bond, change dramatically the conformational properties of studied dehydropeptides. Especially, the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Ac‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuo and in the polar environment. On the contrary, N‐methylated E isomer (Ac‐Gly‐(E)‐ΔPhe‐NMe₂) can easier adopt β‐turn conformation, but the backbone torsion angles (?₁, ψ₁, ?₂, ψ₂) are off the limits for common β‐turn types. © 2012 Wiley Periodicals, Inc. Biopolymers 97:518–528, 2012.     

Elucidating the role of Trp105 in the KPC-2 ��-lactamase

The molecular basis of resistance to β‐lactams and β‐lactam‐β‐lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective β‐lactam therapy. Recent crystal structures of KPC‐2 and other class A β‐lactamases suggest that Ambler position Trp105 may be of importance in binding β‐lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC‐2 by conducting site‐saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, ‐Tyr, ‐Asn, and ‐His KPC‐2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC‐2. Interestingly, most of the variants showed increased MICs to ampicillin‐clavulanic acid but not to ampicillin‐sulbactam or piperacillin‐tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, ‐Asn, ‐Leu, and ‐Val) were studied in detail. Consistent with the MIC data, the Trp105Phe β‐lactamase displayed improved catalytic efficiencies, k_cat/K_m, toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k_cat/K_m toward cefotaxime and imipenem when compared to WT β‐lactamase. The Trp105Asn variant exhibited increased K_ms for all substrates. In contrast, the Trp105Leu and ‐Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k_cat/K_m) for all substrates. With respect to clavulanic acid, the K_is and partition ratios were increased for the Trp105Phe, ‐Asn, and ‐Val variants. We conclude that interactions between Trp105 of KPC‐2 and the β‐lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in β‐lactam and β‐lactamase inhibitor discrimination.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Inter‐subunit interaction of gastric H+,K+‐ATPase prevents reverse reaction of the transport cycle

The gastric H⁺,K⁺‐ATPase is an ATP‐driven proton pump responsible for generating a million‐fold proton gradient across the gastric membrane. We present the structure of gastric H⁺,K⁺‐ATPase at 6.5 Å resolution as determined by electron crystallography of two‐dimensional crystals. The structure shows the catalytic α‐subunit and the non‐catalytic β‐subunit in a pseudo‐E₂P conformation. Different from Na⁺,K⁺‐ATPase, the N‐terminal tail of the β‐subunit is in direct contact with the phosphorylation domain of the α‐subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β‐subunit N‐terminus allowed the reverse reaction to occur. These results suggest that the β‐subunit N‐terminus prevents the reverse reaction from E₂P to E₁P, which is likely to be relevant for the generation of a large H⁺ gradient in vivo situation.     

Crystal structure of agkisacucetin, a Gpib-binding snake C-type lectin that inhibits platelet adhesion and aggregation

Agkisacucetin is a snake C‐type lectin isolated from the venom of Agkistrodon acutus (A. acutus). It binds specifically to the platelet glycoprotein (GP) Ib and prevents the von Willebrand factor (VWF) accessing it. We determined the crystal structure of agkisacucetin to 1.9Å resolution. The structure of agkisacucetin has an (αβ) fold similar to another GPIb‐binding protein, flavocetin‐A, but lacks the C‐terminal cysteine in the β‐subunit, does not form (βα)₄ tetramers, and does not cluster GPIbs, like flavocetin‐A. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Toward engineering efficient peptidomimetics. Screening conformational landscape of two modified dehydroaminoacids

Effective peptidomimetics should posses structural rigidity and appropriate interaction pattern leading to potential spatial and electronic matching to the target receptor site. Rational design of such small bioactive molecules could push chemical synthesis and molecular modeling toward faster progress in medicinal chemistry. Conformational properties of N‐t‐butoxycarbonyl‐glycine‐(E/Z)‐dehydrophenylalanine N′,N′‐dimethylamides (Boc‐Gly‐(E/Z)‐ΔPhe‐NMe₂) in chloroform were studied by NMR and IR spectroscopy. The experimental findings were supported by extensive calculations at DFT(B3LYP, M06‐2X) and MP2 levels of theory and the β‐turn tendency for both isomers of the studied dipeptide were determined in vacuum and in solution. The theoretical data and experimental IR results were used as an additional information for the NMR‐based determination of the detailed solution conformations of the peptides. The obtained results reveal that N‐methylation of C‐terminal amide group changes dramatically the conformational properties of studied dehydropeptides. Theoretical conformational analysis reveals that the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Boc‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuum and in polar environment. On the contrary, N‐methylated E isomer (Boc‐Gly‐(E)‐ΔPhe‐NMe₂) can easily adopt β‐turn conformation, but the backbone torsion angles (φ₁, ψ₁, φ₂, ψ₂) are off the limits for common β‐turn types. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 28–40, 2014.     

Secretory expression of a β‐xylosidase gene from Thermomyces lanuginosus in Escherichia coli and characterization of its recombinant enzyme

Aims: To characterize a β‐xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β‐xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017‐bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β‐xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6‐fold to homogeneity with an estimated mass of 51·6 kDa by SDS‐PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a K_i value of 63 mmol l^?1. Conclusions: In this study, a GH family 43 β‐xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β‐xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.     

Structural insight into the high reduction potentials observed for Fusobacterium nucleatum flavodoxin

Flavodoxins are small flavin mononucleotide (FMN)‐containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate‐binding loop (T/S‐X‐T‐G‐X‐T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The E_ox/sq (oxidized/semiquinone; ?43 mV) and E_sq/hq (semiquinone/hydroquinone; ?256 mV) are the highest recorded reduction potentials of known long‐chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70‐fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive E_ox/sq. The more electropositive E_sq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.     

β‐Adrenergic‐induced CD40 overexpression on gingival fibroblasts: Role of PGE2

CD40, a member of the tumour necrosis factor‐α receptor family, is constitutively expressed by cells of haematopoietic and non‐haematopoietic origin, including fibroblasts. Signalling through this receptor molecule regulates inflammatory mediator secretion by many cell types. The work has been performed in healthy subjects and the authors studied, by cellular culture, flow cytometric analysis and ELISA assay, the expression of CD40 and PGE₂ (prostaglandin E₂) generation on gingival fibroblasts stimulated by β‐AR (β‐adrenoceptor) agonists. Herein, the authors demonstrate that β‐AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by β‐AR subtype‐specific antagonists. In addition, gingival fibroblast β‐AR stimulation resulted in an increase in PGE₂ generation. The inhibition of PLA₂ (phospholipase A₂) and COX‐1 (cyclo‐oxygenase‐1) but not COX‐2 impaired β‐AR increase of PGE₂, an effect that was restored by the addition of low concentrations of PGE₂, suggesting that PGE₂ generation is implicated in the mechanism underlying β‐AR‐agonist‐mediated CD40 overexpression. Our work has revealed an endogenous β‐AR mediator network involving gingival fibroblasts.     

Phospholipase A2 activity of β‐bungarotoxin is essential for induction of cytotoxicity on cerebellar granule neurons

The aim of this study was to investigate the mechanism of the cytotoxic effect of β‐bungarotoxin (β‐BuTX), a presynaptic neurotoxin, on rat cerebellar granule neurons (CGNs). The maturation of CGNs is characterized by the prominent dense neurite networks that became fragmented after treatment with β‐BuTX, and this cytotoxic effect of β‐BuTX on CGNs was in a dose‐ and time‐dependant manner. The cytotoxic effect of β‐BuTX was found to be more potent than other toxins, such as α‐BuTX, cardiotoxin, melittin, and Naja naja atra venom phospholipase A₂. Meanwhile, undifferentiated neuroblastoma neuronal cell lines, IMR‐32 and SK‐N‐MC, and astrocytes were found to be resistant to β‐BuTX. These results indicated that only the mature CGNs were sensitive to β‐BuTX insults. None of the following chemicals: antioxidants, K⁺‐channel activator, K⁺‐channel antagonists, intracellular Ca²⁺ chelator, Ca²⁺‐channel blockers, NMDA receptor antagonists, and nitric oxide synthase inhibitor tested, were able to reduce β‐BuTX‐induced cytotoxicity. However, secretory type phospholipase A₂ inhibitors (glycyrrhizin and aristolochic acid) and a free radical scavenger (5,5‐dimethyl pyrroline N‐oxide, DMPO) could attenuate not only β‐BuTX‐induced cytotoxicity but also ROS production and caspase‐3 activation. These data suggest that phospholipase A₂ activity of β‐BuTX may be responsible for free radical generation and caspase‐3 activation that accounts for the observed cytotoxic effect. It is proposed that the CGNs can be a useful tool for studying interactions of the molecules on neuronal plasma membrane with β‐BuTX that mediates the specific cytotoxicity. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Different contribution of conserved amino acids to the global properties of triosephosphate isomerases

It is generally assumed that the amino acids that exist in all homologous enzymes correspond to residues that participate in catalysis, or that are essential for folding and stability. Although this holds for catalytic residues, the function of conserved noncatalytic residues is not clear. It is not known if such residues are of equal importance and have the same role in different homologous enzymes. In humans, the E104D mutation in triosephosphate isomerase (TIM) is the most frequent mutation in the autosomal diseases named “TPI deficiencies.” We explored if the E104D mutation has the same impact in TIMs from four different organisms (Homo sapiens, Giardia lamblia, Trypanosoma cruzi, and T. brucei). The catalytic properties were not significantly affected by the mutation, but it affected the rate and extent of formation of active dimers from unfolded monomers differently. Scanning calorimetry experiments indicated that the mutation was in all cases destabilizing, but the mutation effect on rates of irreversible denaturation and transition‐state energetics were drastically dependent on the TIM background. For instance, the E104D mutation produce changes in activation energy ranging from 430 kJ mol^?1 in HsTIM to ?78 kJ mol^?1 in TcTIM. Thus, in TIM the role of a conserved noncatalytic residue is drastically dependent on its molecular background. Accordingly, it would seem that because each protein has a particular sequence, and a distinctive set of amino acid interactions, it should be regarded as a unique entity that has evolved for function and stability in the organisms to which it belongs. Proteins 2014; 82:323–335. © 2013 Wiley Periodicals, Inc.     

Oestrogen induces epithelial‐mesenchymal transition in endometriosis via circ_0004712/miR‐148a‐3p sponge function

Endometriosis is a common, chronic gynaecologic disease affecting up to 10% of women in their reproductive age and leading to pain and infertility. Oestrogen (E₂)‐induced epithelial‐mesenchymal transition (EMT) process has been considered as a key factor of endometriosis development. Recently, the dysregulated circular RNAs (circRNAs) have been discovered in endometriosis tissues. However, the molecular mechanism of circRNAs on the E₂‐induced EMT process in endometriosis is still unknown. Here, we demonstrated that circ_0004712 up‐regulated by E₂ treatment in endometrial epithelial cells. Knock‐down the expression of circ_0004712 significantly suppressed E₂‐induced cell migration activity. Meanwhile, we identified miR‐148a‐3p as a potential target miRNA of circ_0004712. Inhibited the expression of miR‐148a‐3p could recovered the effect of circ_0004712 knock‐down in E₂‐treated endometrial epithelial. Furthermore, Western blot assay showed that E₂ treatment could increase the expression and activity of β‐catenin, snail and N‐cadherin and reduce the expression of E‐cadherin. The expression and activity of β‐catenin pathway were recovered by circ_0004712 knock‐down or miR‐148a‐3p overexpression. Altogether, the results demonstrate that circ_0004712/miR‐148a‐3p plays an important role in E₂‐induced EMT process in the development of endometriosis, and the molecular mechanism may be associated with the β‐catenin pathway. This work highlighted the importance of circRNAs in the development of endometriosis and provide a new biomarker for diagnosis and therapies.     

Predicting memapsin 2 (��-secretase) hydrolytic activity

Memapsin 2 (BACE1, β‐secretase), a membrane aspartic protease, functions in the cleavage of brain β‐amyloid precursor protein (APP) leading to the production of β‐amyloid. Because the excess level of β‐amyloid in the brain is a leading factor in Alzheimer's disease (AD), memapsin 2 is a major therapeutic target for inhibitor drugs. The substrate‐binding cleft of memapsin 2 accommodates 12 subsite residues, from P₈ to P₄′. We have determined the hydrolytic preference as relative k_cat/K_M (preference constant) in all 12 subsites and used these data to establish a predictive algorithm for substrate hydrolytic efficiency. Using the sequences from 12 reported memapsin 2 protein substrates, the predicted and experimentally determined preference constants have an excellent correlation coefficient of 0.97. The predictive model indicates that the hydrolytic preference of memapsin 2 is determined mainly by the interaction with six subsites (from P₄ to P₂′), a conclusion supported by the crystal structure B‐factors calculated for the various residues of transition‐state analogs bound to different memapsin 2 subsites. The algorithm also predicted that the replacement of the P₃, P₂, and P₁ subsites of APP from Val, Lys, and Met, respectively, to Ile, Asp, and Phe, respectively, (APP_IDF) would result in a highest hydrolytic rate for β‐amyloid‐generating APP variants. Because more β‐amyloid was produced from cells expressing APP_IDF than those expressing APP with Swedish mutations, this designed APP variant may be useful in new memapsin 2 substrates or transgenic mice for AD studies.     

X-ray evidence of a native state with increased compactness populated by tryptophan-less B. licheniformis β-lactamase

β‐lactamases confer antibiotic resistance, one of the most serious world‐wide health problems, and are an excellent theoretical and experimental model in the study of protein structure, dynamics and evolution. Bacillus licheniformis exo‐small penicillinase (ESP) is a Class‐A β‐lactamase with three tryptophan residues located in the protein core. Here, we report the 1.7‐Å resolution X‐ray structure, catalytic parameters, and thermodynamic stability of ESP^ΔW, an engineered mutant of ESP in which phenylalanine replaces the wild‐type tryptophan residues. The structure revealed no qualitative conformational changes compared with thirteen previously reported structures of B. licheniformis β‐lactamases (RMSD = 0.4–1.2 Å). However, a closer scrutiny showed that the mutations result in an overall more compact structure, with most atoms shifted toward the geometric center of the molecule. Thus, ESP^ΔW has a significantly smaller radius of gyration (R_g) than the other B. licheniformis β‐lactamases characterized so far. Indeed, ESP^ΔW has the smallest R_g among 126 Class‐A β‐lactamases in the Protein Data Bank (PDB). Other measures of compactness, like the number of atoms in fixed volumes and the number and average of noncovalent distances, confirmed the effect. ESP^ΔW proves that the compactness of the native state can be enhanced by protein engineering and establishes a new lower limit to the compactness of the Class‐A β‐lactamase fold. As the condensation achieved by the native state is a paramount notion in protein folding, this result may contribute to a better understanding of how the sequence determines the conformational variability and thermodynamic stability of a given fold.