The ultimate goal in any biosensor development project is its use for actual sample detection. Recently, there has been an interest in biosensors with aptamers as bio-recognition elements, but reported examples all deal with standards, not human serum. In order to verify the differences of aptamer-based biosensor and antibody-based biosensor in clinical detection, a comparison of the performance of aptamer-based and antibody-based quartz crystal microbalance (QCM) biosensors for the detection of immunoglobulin E (IgE) in human serum was carried out. Aptamers (or antibodies) specific to IgE were immobilized on the gold surface of a quartz crystal. The frequency shifts of the QCM were measured. The linear range with the antibody (10–240 μg/L) compared to that of the aptamer (2.5–200 μg/L), but a lower detection limit could be observed in the aptamer-based biosensor. The reproducibility of the two biosensors was comparable. The aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity. In addition, the aptamer receptors could tolerate repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. When stored for three weeks, the frequency shifts of the aptamer-coated crystals were all greater than 90% of those on the response at the first day. The SELEX-derived aptamers generally have a high degree of similarity to antigen–antibody binding in both their specificity and affinity. Aptamers are synthetic, single-stranded DNA or RNA molecules that fold up into unique 3-D structures, which allows them to specifically bind to other target molecules. Since their discovery about 20 years ago ,2, aptamRecently, the stability and convenience of DNA aptamers have been exploited in diagnostic applications including biosensors ,12 and fThe key issue in the development of aptamer-based sensors is to convert target recognition into a measurable signal. QCM crystal transducers are known to be highly precise, stable oscillators and are capable of detecting subnanogram mass changes . SeveralWe have set up a QCM immunosensor platform and successfully quantified human chorionic gonadotropin  and urin2.2.1.We compared the aptamer-based biosensor with the antibody-based biosensor with respect to achieved sensitivity and selectivity. The aptamer and antibody-coated crystals were incubated in IgE solutions in a range from 2.5–250 μg/L, both aptamer and antibody-based crystals showed typical binding capacity saturation. Theaptamer-based biosensor displayed signal saturation at the concentration of 200 μg/L IgE. The antibody-based biosensor performed similarly, but not exhibiting saturation below a concentration of 240 μg/L IgE. Although aptamers were likely to be immobilized in a denser arrangement than antibodies due to their smaller size, signal saturation did not shift to higher concentrations. This effect may be caused by steric hindrance between bound analyte molecules. The antibody-based biosensor generated significantly lower detection signals (ΔF), possibly caused by partial denaturation of the immobilized antibodies on the surface of crystals, leading to a decreasing number of correctly folded antibodies being available for specific analyte recognition.Concerning the limit of detection, aptamers were proved to be superior compared to antibodies. The limit of detection  was measured on 20 consecutive negative controls. The antibody-based biosensor was able to specifically detect IgE at a minimum concentration of 10 μg/L. In addition, specific analyte recognition by the aptamer-based biosensor could be observed down to a concentration of 2.5 μg/L in the binding assay. This result most likely reflected the dense and highly ordered nature of the aptamer receptor layer. The reaction time to reach equilibrium for both biosensors was 15 min. In a previous approach, anti-IgE antibodies and aptamers were compared as receptor molecules using a quartz crystal microbalance biosensor. Both receptor types detected IgE specifically at a minimum concentration of 95 μg/L [2.2.Imprecision data for the determination of IgE (2.5–200 μg/L) by the aptamer or antibody-based biosensor was compared intraassay and interassay. For every concentration, tests were repeated 20 times in one day for intraassay and repeated on 20 consecutive days in the same manner (mean of three duplicates per day) for interassay reproducibility. The mean intraassay and interassay CV of aptamer-based biosensor were 4.14% and 5.95%, respectively. Similarly, the intraassay and interassay CV of the antibody-based biosensor were 4.18% and 6.13%. Variable surface coverage between manually produced sensing elements might account for this precision difference. Large-scale, automated fabrication of aptamer biosensors would likely yield much more uniform surface coverage and a correspondingly lower CV.2.3.We further tried IgE detection in human serum containing a variety of proteins, including different types of immunoglobulins. IgE concentrations in clinical human serum samples were simultaneously measured by the QCM biosensor and the chemoluminescence method. Mean values by the aptamer-based QCM biosensor, antibody-based QCM biosensor and chemoluminescence in 50 clinical human serum samples were 64.0, 62.6 and 64.9 μg/L, respectively, with ranges of 3–215, 5–234 and 5–208 μg/L. To investigate the correlation of the QCM biosensor with the chemoluminescence method, the Bland-Altman difference plot analysis for the clinical sample detection results was done . A Bland2.4.One of the greatest challenges a protein biosensor faces is nonspecific binding, which results in high background signals and therefore decreasing assay sensitivity. In this respect, a comparison between the signal intensities of specific IgE recognition with nonspecific protein binding again emphasized the superiority of aptamers. To test the specificity of the aptamer, proteins with different molecular weight and isoelectric point were used. The interfering proteins that were tested included HSA, lysozyme, IgG, and IgM. These proteins were chosen to cover the range of possible interfering molecules in the detection of IgE. HSA has a much smaller molecular weight  with respect to IgE , whereas IgG is structurally similar to IgE and thus can serve as a good test protein for the aptamer binding specificity. The concentration of the interfering proteins was chosen to be 500 mg/L; it was a very high concentration compared with the tested IgE. The relative responses of the interferences, as seen in 2.5.The regeneration of binding surfaces is important for reusable biosensors but it often difficult to achieve. We tried to regenerate the biosensor surface after binding of IgE by rinsing the antibody-coated crystals with 0.2 mmol/L glycine/HCl (pH 2.8), or 1.2 mmol/L NaOH, the common regents for dissociating antibody-antigen complexes. This resulted in a complete release of the analyte. However, all subsequent injection of IgE only led to reduced frequency changes. Apparently, the receptor layer was either irreversibly damaged or the antibodies were unfolded. The refolding of the antibodies was too inefficient to be effective before subsequent injection of the analyte. Although the antibody-based biosensor could be reusable, a loss of activity of the immobilized antibody was inevitable.2+-containing buffer [Unlike the antibody-based biosensor, the aptamer-based biosensor could be denatured and reused many times on the same chip, without loss of function. Binding of IgE to the aptamers was an almost completely reversible procedure. As shown in g buffer .Aptamer or antibody-coated crystals were covered with 0.02% sodium azide solution in PBS and stored at 4 °C for long-time stability test. We made three different crystals for measurement per day continuously for 35 days. At the end of the third week, the loss of adsorbed surface mass was calculated to be only 10%, indicating loss of probes was negligible over this time period for aptamer-coated crystals. For antibody-coated crystals, the stability was almost fully maintained at the first two days and then started to decline. After one week, the relative frequency shifts of antibody-coated crystals were about 40% compared with its initial response. This is because that the antibodies are sensitive to temperature and undergo irreversible denaturation. The antibodies also have a limited shelf life. The reusability and long-time stability of the aptamer-based biosensor showed that the cost of detection could be reduced by this method.3.3.1.N-succinimidyl ester) (DSP), N,N-dimethylacetamide (DMA), staphylococcal protein A (SPA), and lysozyme were purchased from Sigma . Bovine serum albumins (BSA) and human serum albumins (HSA) were purchased from Roche . IgE, IgG and IgM were purchased from US Biological Company . All the other reagents were purchased from Sigma . Monoclonal anti-human IgE antibody was purchased from Beckman . The composition of the buffers used for the experiments is reported below:Immobilization buffer: 10 μM Tris-HCl buffer, 0.2 M NaCl, pH 7.9.2HPO4, 1.1 mM KH2PO4, 1 mM MgCl2, 2.7 mM KCl, and 138 mM NaCl, the pH was adjusted to 7.4, which was based on the reported SELEX conditions [Reaction buffer: 8.1 mM Nanditions .Avidin, 3,3′-dithiodipropionic acid di(The base sequence for the anti-IgE aptamer (D17.4) has been found to be 5′-GGGGCACGTTTAT-CCGTCCCTCCTAGTGGCGTGCCCC-3′, as noted from the initial work of Wiegand and co-workers . The apt3.2.2O2: 98% H2SO4 = 1:3) and then thoroughly washed with tri-distilled water and dried with pure nitrogen gas.The 10 MHz AT cut quartz crystal (13 mm × 13 mm) with gold evaporated on both sides was obtained from 26th Research Institute, Chinese Electronic Scientific and Technical Group Company . The 2 × 5 model micro-array sensor was made by China Jialing Group . The frequency variations were continuously recorded using a frequency data acquisition card : the data (the resonance frequency) was displayed on the display screen and could be read directly by a computer connected to the PCL-836 interface. The frequency shifts reported were the difference between two stable frequency values (±1 Hz). The cell was used in static conditions. Temperature was controlled (≤1 °C) by an air thermostat. The measuring apparatus consisted of the crystals fixed inside the sensor, only one side of which was exposed to the solutions. All detection wells were designed with the same structure, and each of them was driven by an independent logic circuit so that they worked independently, without mutual interference. The electrode surface was soaked and rinsed for 10 min in cleaning solution  was monitored until a steady baseline was obtained. Then 10 μL IgE standards of different concentrations (2.5–250 μg/L) were added into the wells respectively. PBS buffer was added to chamber 10 as a negative control. When the resonance frequency reach a stable value, that means the reaction between the aptamer (or antibody) and the IgE came to an end. Then, another steady state resonant frequency was taken as F1. The frequency shifts (ΔF = F1 – F0) induced by aptamer-IgE (or antibody-IgE) were the difference between the final value (F1) and the value displayed before reaction (F0). The equilibrium time for reaction was also recorded. The average value was taken by repeating the experiment 3 times. The dose-curve was drawn according to the relationship between frequency shifts and IgE concentrations. Human IgG, IgM, and HSA (Sigma) were used as nonspecific binding controls. All proteins were dissolved in a PBS buffer at a concentration of 500 mg/mL, which served as a stock solution.Ten prepared crystals were placed into the detection wells with the side coated with aptamer (or antibody) facing up to construct the 2 × 5 model array. The system temperature was adjusted to 37 °C. Then, 90 μL of reaction buffer was added to each cell of the sensor. The resonance frequency , 1.2 mmol/L NaOH, and 30 mmol/L EDTA, were applied on the electrodes for two minutes to denature the aptamer or antibody and remove the IgE from it after each measurement. The obtained surfaces were then rinsed with PBS buffer three times. After washing with tri-distilled water the frequency reached a stable value (±1 Hz) with buffer, aptamer or antibody once again responded to the IgE and a new detection step could be performed.3.7.First, we generated the calibration curves by plotting ΔF against various IgE concentrations, and the linearity was determined by serially diluting the stock IgE solutions. After the analysis procedures, the operational characteristics, including imprecision, accuracy, specificity, analytical sensitivity, and detection limit, were evaluated. Then, the serum samples were quantified individually with the QCM biosensor and chemiluminescence . All participants gave signed informed consent and the study was approved by the Ethics Boards of the Third Military Medical University. Bland-Altman analyses were performed to evaluate the residuals plots of the proposed methods and reference method.3.8.t-test, and the Bland-Altman analyses were performed with GraphPad, version 5.0.The results are expressed as mean (±SD). SPSS 10.0 software was used for the 4.Antibodies and aptamers can bind to their targets with high affinity and specificity, aptamers offer distinct advantages over antibodies that make them very promising in analytical and diagnostic applications. These advantages include the easily regenerating the function of the immobilized aptamers and the possibility of using different detection methods due to easy labeling ,33. Due Here we assessed a DNA-aptamer as an affinity ligand, and it compared favorably with its antibody counterpart. Compared to antibodies (10 ng/mL), aptamer-based analyte recognition was at least as sensitive. Aptamers could detect their analytes (IgE) at a minimum concentration of 2.5 ng/mL. Regarding specificity, aptamers were even superior to antibodies, which exhibited higher signal backgrounds due to a higher degree of nonspecific protein binding. Moreover, for clinical sample detection, the consistency between QCM aptamer biosensor and chemiluminescence method is good. Another advantage over antibodies can be seen in the higher stability of aptamers, as aptamers are very stable and they can recover their native active conformation after denaturation. The results suggested that the aptamer-based biosensor was sensitive, specific, and easy-operating method. These results raise the prospect that aptamers could be adapted to function as reusable, long-time biosensors in arrays.Recently, there has been an interest in biosensors with aptamers as the bio-recognition element. In most cases, only limited quantities of IgE from standards are available by QCM biosensor. To achieve the object of detecting IgE in clinical serum samples, we combined QCM and aptamer together. QCM has high sensitivity and aptamer has high selectivity. Combining the two elements together, sensitivity was greatly enhanced, and detection can be performed directly on clinical serum samples. Our results showed that this system can detect about 2.5 ng/mL IgE, making it a feasible assay for practical applications. In conclusion, this aptamer-based QCM biosensor can directly detect IgE from clinical serum samples with high sensitivity and selectivity. It may be a rapid, efficient and simple approach for detection of IgE in clinical specimens. Given that QCM immunosensor analyses are fairly rapid and relatively easy to operate, this approach lends itself particularly well to the rapid development of an analytical method in clinical laboratories.


HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1. Complete eradication of HIV-1 infection requires novel approaches to induce integrated HIV-1 provirus which is not affected by the existing antiretroviral drugs and which is rebound upon the termination of the antiretroviral therapy . Thus, b1YSPTSPS7 heptapeptide sequence repeated 52 times in the C-terminal domain (CTD) of RNAPII [HIV-1 transcription is activated by HIV-1 Tat protein that binds to the bulge of TAR RNA, a hairpin-loop structure located at the 5′-end of all nascent HIV-1 transcripts, and recruits CDK9/cyclin T1, a component of positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter  and one of the C-type cyclins T1, T2a, or T2b of which cyclin T1 is the most abundant partner , 13. CycThere are two functional isoforms of CDK9: a 42 kDa form predominately expressed in spleen, thymus, and testis and a 55 kDa form that is expressed in various tissues but at significantly lower levels that is the 42 Kd isoform , 22. Allin vitro, the large complex, and not the small complex, is important for the activation of HIV-1 transcription as HIV-1. Tat competes with HEXIM1 for binding to cyclin T1 promoting dissociation of P-TEFb from the large complex [The high molecular weight P-TEFb complex serves as a source of CDK9/cyclin T1 for the recruitment by HIV-1 Tat . In stre complex , 39. HIV complex .Tat also facilitates the formation of super elongation complex (SEC) containing active P-TEFb and additional elongation factors and transcription coactivators , 42. TheActivity of P-TEFb is dependent upon phosphorylation of several serine and threonine residues and we will discuss this below and which are shown in the CDK9/cyclin T1/Tat model in in vitro was efficiently dephoshorylated by protein phosphatase 2A (PP2A) but not the protein phosphatase-1 (PP1) [in vitro [in vitro was inhibited by TFIIH [in vitro, as PP2A depletion inhibited basal HIV-1 transcription and the add-back of PP2A to the depleted extracts restored the transcription [in vivo. In an early study, we observed a mild induction of basal but not Tat-induced HIV-1 transcription by PP2A-inhibitory low concentrations of okadaic acid [in vitro [A cluster of CDK9's C-terminal residues  is autophosphorylated and this phosphorylation is important for the binding of CDK9/cyclin T1 to TAR RNA , 45, 46.by TFIIH . PP2A wacription . The PP2in vitro . CollectPhosphorylation of CDK9's Thr-29 was shown to be induced by HTLV-1 Tax protein . CDK9/cyCDK9 T loop contains several phosphorylation sites, including Ser-175 and Thr-186 . Phosphoin vivo [Recently, chemical-genetic analysis using selective chemical inhibition of CDK7 showed that it is solely responsible for CDK9 Thr-186 phosphorylation and for P-TEFb-dependent CTD Ser-2 phosphorylation and histone H2B ubiquitylation in vivo . Thus, Cin vivo ). Globalin vivo . Accordiin vivo . Becausein vitro [in vitro and in vivo and that mutation of residues in the PP1 binding motif (V36A and F38A) prevented Tat from inducing HIV-1 transcription [32P)orthophosphate and treated with okadaic acid was efficiently dephophosphorylated in vitro by PP1 but not PP2A [in vitro autophosphorylated CDK9 that was dephosphorylated by PP2A and not by PP1 [α, was shown to act cooperatively and sequentially with (Ca2+)-calmodulin-protein phosphatase 2B (PP2B) in UV irradiated or hexamethylene bisacetamide (HMBA)-treated cells in which P-TEFb is released from 7SK snRNP complex [α dephosphorylates CDK9 Thr-186 and facilitates the releases of P-TEFb from 7SK snRNP [nef potently inhibited HIV-1 replication [CDK9 Thr-186 phosphorylation can also be controlled by cellular phosphatases. Our studies for the past decade focused on PP1 that we initially observed to stimulate Tat-dependent HIV-1 transcription in vitro . When wein vitro . We noticription . CDK9 thnot PP2A  in contrt by PP1 . These r complex . While PSK snRNP . CDK9 reSK snRNP . Stable SK snRNP , suggestlication , furtherlication . PPM1A olication . A more lication . This filication , yet unkin vivo [in vitro and in vivo [Once CDK9/cyclin T1 dissociates from the large P-TEFb complex, CDK9 may become phosphorylated on Ser-175. While Thr-186 dephosphorylation totally inhibited CDK9/cyclin T1 kinase activity, no difference was found in the kinase activities of CDK9 S175A and CDK9 S175D mutants by David Price and colleagues . In contin vivo . Inhibit in vivo . We foun in vivo . Interes in vivo . Based o in vivo . Also in in vivo . Also, i in vivo . They al in vivo , which p in vivo . Thus, tin vitro, disrupted the association of PP1 with Tat in cultured cells, and prevented the translocation of PP1 to the nucleus [We recently developed PP1-targeted small molecules that were modeled to fit the RVxF-accommodating cavity on PP1 . We scre nucleus . We are 311 and ICL670 which also inhibited the cellular activity of CDK2 and CDK9 [α (HIF-1α) as iron depletion removes iron from prolyl hydroxylase and increases HIF-1α and HIF-2α protein levels mimicking the effect of hypoxia [in vitro and identified a motif (90SPYNR94) that represented a consensus CDK2 phosphorylation site and which was efficiently phosphorylated [We and others have shown earlier that HIV-1 transcription is activated by Tat in the G1 phase, but not in the G2 phase , 72. Thuand CDK9 . More poand CDK9 . While t hypoxia . We analorylated . Phosphoorylated  suggestiPhosphorylation and dephosphorylation of specific sites on CDK9 occurs throughout the transcription process and must be tightly controlled. Modifications at certain threonine/serine residues determine whether CDK9 associates with large P-TEFb complex to become available for recruitment and whether the dissociation of the large complex will efficiently occur. Phosphorylation of Thr-186 in the T-loop of CDK9 and Ser-90 which lies outside of the T-loop determines whether CDK9 remains sequestered in the inactive large complex and also whether the kinase is enzymatically active once it is dissociated from 7SK snRNP. Dephosphorylation at either of these sites leads to destabilization of the large complex and release of CDK9/Cyclin T1, thereby restricting Tat recruitment to HIV-1 LTR. Modifications in the CDK9's C-terminus allow for cyclin T1 : TAR binding and dephosphorylation at these sites prevents binding toTAR RNA. In contrast, phosphorylation at Thr-29 or Ser-175 residues inhibits CDK9. Thus the emerging picture of CDK9 regulation through phosphorylation seems to be complex and in part parallels what is known for other CDKs. The recently identified CDK9-targeted kinases, CDK7, CDK2, and phosphatases, PP1, and PPM1A will likely emerge as novel targets for anti-HIV-1 and cancer therapeutics.


The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold  exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. The remarkable ability of enzymes to selectively catalyze the reactions of compounds from the cellular soup is essential for the proper functioning of most pathways in biological systems A quantitative measure of broad substrate specificity is yet to be formalized. A previous attempt to quantify broad substrate specificity provided a measure of the catalytic efficiencies of an enzyme toward a pre-defined set of substrates, but was limited in its scope and scalability Broad substrate specificity estimator (BRASS). BRASS computes ‘duplicate’ residues from the structure of a protein with known active site residues. These ‘duplicate’ residues generate slightly modified replicas of the active site scaffold. Finite difference Poisson Boltzmann analysis is used to filter out electrostatically unfavorable configurations In the current work, we quantitatively characterize the active site of an enzyme to measure broad substrate specificity - duplicate’ residues can be subjected to saturation mutagenesis which selects for high specificity with respect to a desired substrate We identified an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of proteins extracted from the Catalytic Site Atlas The critical role of the ribosome as the site for protein synthesis in cell viability makes it the logical target for a wide range of drugs Palmitoylation is a critical protein modification that governs protein-protein interactions, protein trafficking and membrane localization Moreover, PPT1 is known to have a much broader substrate profile than a related thioesterase (PPT2), with which it shares 26% identity Platelet-activating factor (PAF) is a very potent messenger phospholipid, found in picomolar concentrations in fluids like cytosol, blood plasma and urine CLASP provides the flexibility in the search process by allowing a user to provide a defined set of residues to match a particular position of the active site motif. Furthermore, the search algorithm is parameterized to exclude matches if any pairwise distance deviation exceeds a user specified threshold. In the default mode, CLASP scores any pairwise deviation more than 2 Å in distance highly, thus eliminating that match as a significant one. We relaxed the spatial constraint, increasing this threshold distance to 5 Å. We created the following groups - BASIC = [Lys His Arg], ACIDIC = [Glu Asp], AMIDE = [Asn Gln], NONPOLAR = [Gly Ala Val Leu Ile Met], AROMATIC = [Phe Trp Tyr], and applied the BRASS algorithm to the chosen set of proteins under consideration.The more flexible search by BRASS identified a ketosteroid isomerase (KSI) (PDBid:1C7H), which catalyzes an allylic isomerization reaction at a diffusion-controlled rate, with a high BrassIndex. The KSI uses a network of hydrogen bonds to connect the critical residues Tyr16 and Asp103. Apart from these two critical residues, Asp40 and Val104 constituted the active site motif as specified in the CSA database . The non-critical residues were allowed to be substituted by stereochemically equivalent residues, i.e. Asp104 and Val104 could be replaced by the elements of the groups BASIC and NONPOLAR respectively. Vibrio alkaline phosphatase (VAP) (PDBid: 3E2D), there are three metal ions similar to most other alkaline phosphatases - the M1 site  binds a zinc ion, the M2 site  binds another zinc ion, while the M3 site  binds a magnesium ion Zinc binding sites are classified as catalytic, structural or cocatalytic in vitro measure of these characteristics due to the laborious work involved, and the huge number of enzymes being discovered in silico methodologies to address this requirement with predictive models for some of these characteristics Evolution has shaped enzymes in accordance to their niche in the cell. Varied physiological constraints have molded enzymes to be mostly efficient Broad substrate specificity estimator (BRASS). We hypothesize that ‘duplicate’ residues generate slightly modified, both spatially and electrostatically, replicas of the active site scaffold that are responsible for a broad substrate profile. Finite difference Poisson Boltzmann analysis is used as a discriminator to rule out electrostatically unfavorable configurations In the current work, we quantitatively characterize the active site of an enzyme to measure broad substrate specificity - BRASS was applied to a set of non-homologous proteins extracted from the Catalytic Site Atlas de novo approaches Thus, BrassIndex applies to a particular enzyme, and is not a characteristic of the class of enzymatic reactions. Directed evolution is a generic term for methods that mimic and accelerate evolution The mechanisms underlying the broad specificities possessed by some enzymes have been the subject of intense research. Molecular dynamics simulations have been used to study the basis of the broad substrate profile of cytochrome P450 (CYP) 3A4, which catalyzes the oxidative degradation of a wide range of compounds Staphylococcus aureus have evolved a PBP (PBP2a) with low affinity for β-lactams The physiological needs that modulate the specificities of enzymes might be understood by a study of the evolutionary related penicillin binding proteins (PBP) and serine β-lactamases, where a nucleophilic serine in the conserved SXXK motif forms an acyl-complex with β-lactam antibiotics BRASS essentially works on a static model. However, it implicitly includes dynamics based on the ‘flexibility’ of the main-chain scaffold and mobility of selected side chains inside prearranged folds or ensembles of conformation. The reason for ‘good’ binding or improved selectivity may be distal to the prime residues in the active site. These residues are considered by allowing for spatial variability, of course under the assumption that the distant residues primarily reinforce what their closer counterparts do by allowing for spatial variability. In alkaline phosphatases, residues in the secondary valence shell around the metal ions have effect on catalysis, e.q. by selecting magnesium in the M3-site and keeping correct coordination for catalysis  A caveat in the computation of an index like the BrassIndex is the reliability of the manual step in specifying the set of residues involved in the catalytic reaction. It is not possible to computationally figure out whether two closely placed residues are both essential or redundant for catalysis. In spite of this inevitable source of error, the number of residues that have proximal ‘duplicate’ candidates is a good benchmark for estimating BrassIndex. Electrostatic analysis of the congruence of the specified active site motif with the other ‘replicas’ is another metric used as a discriminator. It should be mentioned that BRASS is unable to detect if and how an enzyme has a specific stereospecificity or steroselectivity, and is simply guided by the specified active site residues. In summary, we quantitatively characterize the properties of an enzyme which results in broad substrate specificity, using spatial and electrostatic properties of residues in the active site and its close vicinity.http://www.pymol.org/).Adaptive Poisson-Boltzmann Solver (APBS) and PDB2PQR packages were used to calculate the potential difference between the reactive atoms of the corresponding proteins The underlying theoretical foundation for CLASP is the non-triviality of the spatial and electrostatic congruence in cognate pairs seen across various structures with the same catalytic mechanism. i)  . All seti)  and 3 ari) . The CLA1 represents the specified active site scaffold of the protein, and has a CLASP score of zero. All matches below a user defined threshold score (Sthresh) are discarded. The index for the degree of specificity should encapsulate the number of scaffolds that are similar to the original active site, as well as the quality of the matches which are reflected by the CLASP scores (2)www.sanchak.com/brass.html.An inherent limitation of CLASP, and typical of other methods that use RMSD, is its inability to distinguish between mirror images. Thus, permutations of the original active site are also excluded. We compute the BrassIndex for a non-homologous set of proteins with known active site and structure extracted from the CSA database CSA provides catalytic residue annotation for enzymes in the PDB and is available online Table S1BRASS results for Cytochrome P450 3A4 (PDBid:3UA1): A flexible loop (residues 211–218) is proposed to be the reason for the broad substrate specificity . BRASS identifies four possibilities for the torsional position of Phe215, which is shown to have the maximum shift upon substrate binding. The core catalytic residues are - Thr309, Glu308, Phe435 and Cys442.(PDF)Click here for additional data file.Table S2Potential and spatial congruence of the active site residues in serine proteases: Chymotrypsin and subtilisin are a classical example of convergent evolution where the catalytic Ser-His-Asp triad shows virtually similar geometry in the structurally different proteins. D = Pairwise distance in Å. PD = Pairwise potential difference. See Methods section for units of potential.(PDF)Click here for additional data file.Table S3Set of non-homologous proteins with known active site residues: The proteins are ordered in an ascending order with respect to the BrassIndex.(PDF)Click here for additional data file.


Ordered progression of mitosis requires precise control in abundance of mitotic regulators. The anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role by directing ubiquitin-mediated destruction of targets in a temporally and spatially defined manner. Specificity in APC/C targeting is conferred through recognition of substrate D-box and KEN degrons, while the specificity of ubiquitination sites, as another possible regulated dimension, has not yet been explored. Here, we present the first analysis of ubiquitination sites in the APC/C substrate ubiquitome. We show that KEN is a preferred ubiquitin acceptor in APC/C substrates and that acceptor sites are enriched in predicted disordered regions and flanked by serine residues. Our experimental data confirm a role for the KEN lysine as an ubiquitin acceptor contributing to substrate destruction during mitotic progression. Using Aurora A and Nek2 kinases as examples, we show that phosphorylation on the flanking serine residue could directly regulate ubiquitination and subsequent degradation of substrates. We propose a novel layer of regulation in substrate ubiquitination, via phosphorylation adjacent to the KEN motif, in APC/C-mediated targeting. Cyclin-dependent kinases (CDKs) act as the hub of this network, with help from other major mitotic kinases such as polo-like kinases (Plks) and Aurora kinases . They usThe process of ubiquitination involves a three-enzyme cascade, where a free ubiquitin is first charged by an E1 activating enzyme and subsequently transferred to an E2 conjugating enzyme. In the presence of an E3 ligating enzyme, which binds both the E2 and the substrate, the ubiquitin is finally used to modify a substrate lysine residue. Importantly, one ubiquitin can modify a second ubiquitin molecule, resulting in eight polyubiquitin chain types with distinct structural topologies and cellular functions . In rapiSurprisingly, among more than 600 E3s in human cells, the anaphase promoting complex/cyclosome (APC/C) is responsible for all the known degradation events controlling mitotic exit . APC/C hin vitro systems.The APC/C uses two specific E2 enzymes for substrate ubiquitination, UBE2C for initial conjugation of one (‘priming’) ubiquitin molecule to substrates and UBE2S to build a polyubiquitin chain on this ubiquitin molecule –16. UBE2in vivo ubiquitination sites [Several recent mass spectrometry-based ubiquitome studies have identified large numbers of on sites –25. This3.3.1.in vivo ubiquitome datasets from several previous studies . We extracted all the ubiquitination sites of APC/C substrates from the non-redundant compendium (see 3.2.a) [p-value = 3.6 × 10–9, binomial test; figure 2b).Previous studies of APC/C substrates have shown that degrons are often found in generally unstructured regions (reviewed in ). We thefigure 2a) ,23,25. IAs the ubiquitin ligase that is responsible for all the known targeted proteolysis in mitotic exit, APC/C has to bind different substrates with high specificity, and yet with considerable flexibility to allow efficient elongation of the ubiquitin chain. The dynamic properties of disordered regions make them good structural candidates for mediating this process, and may explain the enrichment of ubiquitination sites in APC/C substrate disordered regions. Alternatively, our finding may reflect the requirement for an unfolded region in substrate proteins to initiate proteolysis at the proteasome (reviewed in ). Finall3.3.a). This analysis revealed distinct preferences for certain residues in the flanking regions of APC/C ubiquitination sites. The strongest preference was for serine residues in the entire flanking region (figure 3a), suggesting that phosphorylation on these residues could interplay with APC/C-dependent ubiquitination. Indeed, 28% of these serines have been experimentally identified as phosphorylation sites [p-value = 8 × 10–4, binomial test). This enrichment in flanking serines was most significant at the −1 position immediately flanking the ubiquitinated lysine residue . The general preference for lysines in APC/C ubiquitination site flanking regions is consistent with the observation that APC/C substrates are frequently ubiquitinated on multiple sites in the vicinity of degrons [0EN as a preferred ubiquitin chain acceptor in APC/C substrates (figure 3a). This preference was even stronger if we refined the ubiquitination sites in APC/C substrates to those within 40 residues downstream of potential degrons , where APC/C-dependent ubiquitination sites are more likely to be found, based on the known spatial layout of the substrate-recognition catalytic module [figure 3b).We set out to investigate whether there might be common features around the ubiquitinated lysines of APC/C substrates that could function to promote ubiquitination or proteolysis. Different ubiquitome studies have shown that compared with all lysines in the human proteome, ubiquitination sites tend to locate in non-basic environments, although no specific motif was detected ,23. Howefigure 3on sites , a signi degrons . Of partc module . None ofc module  generate3.4.(?P)-K0-E-N) that we had identified, we first searched for APC/C substrates containing an SKEN motif, to examine whether the motif can function as a ubiquitin chain acceptor in vivo. Eight validated APC/C substrates from our list  contain SKEN, three of these  being known sites of ubiquitination . We selected two substrates, hsAurora A and hsNek2A kinases, for in vivo study. We have previously shown that Aurora A is specifically targeted by APC/CCdh1 for degradation in mitotic exit to promote assembly of a robust spindle midzone [To experimentally confirm the ubiquitination site flanking features  [Cdh1-dependent polyubiquitin conjugates at far higher levels than the wild-type protein . We purified His6-Aurora A Δ32–66 from cells synchronized at mitotic exit when Aurora A is maximally ubiquitinated [figure 3c and see electronic supplementary material, figure S3 for details). We concluded that SK5EN is an important site of mitotic ubiquitination in Aurora A.We first examined whether SK A, K250 , was pre degron) , a versi3.5.5EN to ubiquitination and degradation of wild-type Aurora A, we substituted the lysine for arginine (K5R) in full-length Aurora A. Venus-tagged Aurora A-K5R showed identical localization to wild-type Aurora A-Venus (a). To compare the ubiquitination capacity of these two proteins in vivo, we expressed them in a human U2OS cell line [b,c). We confirmed that the K5-dependent ubiquitination of Aurora A contributes to its mitotic destruction by measuring Aurora A proteolysis during time-lapse imaging of live cells [d). Taken together, our data suggest that K5 in the SKEN motif is a major ubiquitination site mediating Aurora A degradation during mitotic exit. We cannot exclude that the KEN motif also contributes to Aurora A ubiquitination through its potential function as a degron, Indeed we have found that the triply substituted KEN > AAA shows a reduced rate of degradation during mitotic exit compared with the version where the lysine residue alone is substituted, K5 > R . It seems likely therefore that the KEN motif plays a dual role in binding to the APC/C and accepting ubiquitin during Aurora A degradation. We tested the doubly substituted KEN > KAA in order to examine further whether K5 plays a more significant role as a ubiquitin acceptor or as part of a degron, but this version of Aurora A (unlike KEN > AAA) did not behave like the wild-type protein in mitotic cells  and was not suitable for further analysis.To test the contribution of SK A-Venus a. To comell line  also expell line  and syncve cells . Quantif3.6.a and electronic supplementary material, figure S2). As serine S4 is a known phosphorylation site on Aurora A in vivo [4KEN. We found that the phospho-mimetic version AurA-S4D-Venus showed increased ubiquitination, as well as a higher maximum degradation rate in mitotic exit, compared with AurA-WT-Venus (b–e). Conversely, the non-phosphorylable version AurA-S4A-Venus showed reduced ubiquitination and a reduced rate of degradation during mitotic exit. The increased ubiquitination and degradation rate of AurA-S4D was fully reversed by additional mutation of K5 . Together, these results suggest that the phosphorylation status of S4 would regulate the ubiquitination efficiency, or the level of ubiquitin occupancy, on K5 of Aurora A.A large number of proteins are highly phosphorylated at mitosis, and phosphate groups on modified serines in disordered regions could provide a negatively charged environment to promote the efficiency of ubiquitination of APC/C substrates . This arfigure 3 in vivo , we examWT-Venus b–e. Conv391EN motif of Nek2A could mediate a similar response to phosphorylation status of the –1 serine. Nek2A degradation depends on its carboxy-terminal methionine–arginine dipeptide tail to mediate binding to APC/C core subunits during spindle checkpoint (SAC)-independent degradation at prometaphase, in combination with its dimerization via a leucine zipper [in vivo [in vivo [f). Meanwhile, the maximum degradation rate was significantly increased by a phospho-mimetic substitution S390D and reduced by non-phosphorylable S390A (f). In agreement with our observations made with Aurora A, phosphorylation of SKEN can likely act to promote ubiquitination at K391 of Nek2A. Nek2A S390 is not a listed phosphorylation site in existing phospho-proteome studies, but is a potential site for CK1 phosphorylation .In summary, we validated our finding that APC/C substrates show sequence preferences in the flanking regions of ubiquitin acceptor sites, by demonstrating that the SKEN motif provides such a site, where the occupancy of the acceptor could be promoted by phosphorylation on the –1 serine residue.4.in vivo and that this ubiquitination event may be regulated at least partly by phosphorylation on flanking serine.In this study, we have found specific preferences in ubiquitination site selection in APC/C substrates, with particular preferences for disordered regions and those enriched in serine residues. We have furthermore demonstrated that the KEN box can be ubiquitinated in vivo.Our identification of APC/C-dependent ubiquitination preferences is necessarily limited firstly because it is not possible to know which sites identified by mass spectrometry in known APC/C substrates are in fact APC/C-dependent, and secondly because APC/C-dependent ubiquitination events leading to rapid degradation of those substrates might be underrepresented in mass spectrometry studies compared with ubiquitination events leading to slower/no degradation. Nonetheless, we were able to extract statistically significant preferences, which could be reinforced by refining our analysis to ubiquitination sites close to APC/C degron motifs, and that we subsequently confirmed in vivo assays for binding of APC/C to its substrates in order to test the relative contributions of KEN binding and ubiquitination to substrate degradation in vivo. Nonetheless, our data suggest that the contribution of the KEN motif to substrate degradation is more complex than existing models allow for.Our finding that the KEN motif can be ubiquitinated seems at first sight curious given the well-described role of KEN as an APC/C-specific degron motif. It raises the possibility that the degron function of the KEN motif could be regulated by ubiquitination. Moreover, it has repeatedly been found that inhibition of the APC/C by pseudo-substrates critically depends on KEN motifs –53, consFrom our finding that serines are preferred in the flanking region of APC/C ubiquitination sites we hypothesized that APC/C substrates would show phosphorylation-mediated regulation of their ubiquitination. Indeed, there are several known examples where APC/C-mediated substrate degradation is regulated by phosphorylation, including that of key substrates securin, Aurora A and Cdc6 –56. HoweaK, enhancing deprotonation. This promotes nucleophilic attack by acceptor lysine on the thioester bond at physiological pH [0EN. A large number of proteins are highly phosphorylated at mitosis, and phosphate groups on modified serines in disordered regions could provide a negatively charged environment to promote the efficiency of ubiquitination of APC/C substrates. Pathways controlling SKEN phosphorylation status have not yet been described. Nonetheless, the particular enrichment of serine over threonine in our analysis suggests that these phosphorylation events could be highly specific.The transfer of ubiquitin from E2 to substrate requires nucleophilic activation of a substrate lysine for attack on the E2-ubiquitin thioester bond. Much of our insight into the mechanism of catalysis comes from studies of lysine modification with SUMO, an ubiquitin-like modifier. Ubc9, an E2 for SUMOylation, provides a hydrophobic microenvironment in its active site that desolvates the acceptor lysine to lower its effective pgical pH . By the gical pH ,31 and p(P)-K-E-N could behave as an efficient motif for ubiquitin chain modifications in promoting mitotic degradation of both Aurora A and Nek2A, supporting our computational analysis showing preferential ubiquitination on lysines flanked by phosphorylatable serines in APC/C substrates. This is the first time it has been shown that the APC/C shows preferences for ubiquitin acceptor sites in any specific sequence context, and that this preference could influence ubiquitination efficiency on APC/C substrates downstream of degron recognition. This preference is likely to contribute to the many dimensions of regulation that control polyubiquitinated substrates ready for mitotic destruction.Together, our experiments show that S5.5.1.Ubiquitination site data were obtained from existing studies –25. Prot5.2.The intrinsic disordered state was predicted for each ubiquitination site using DISOPRED with standard settings . Using tp-values from binomial tests were ln-transformed and plotted as a heat map using the R environment [To identify amino acids that were enriched or depleted in the flanking regions of ubiquitination sites, we extracted ±10 residues of each site in the compendium. For each amino acid at each position relative to the ubiquitination site, we tested the null hypothesis that the frequency of observing this amino acid is not greater  or not less  in the APC/C substrates than in all ubiquitinated proteins, against the alternative hypothesis that it is. ironment  and the ironment . Statistironment .5.3.6-tagged Aurora AΔ32–66 was expressed using pcDNA3.1(+)/myc-His C vector (Invitrogen).HisVenus was swapped for fluorescent proteins in pEYP-N1-AurA  and pEGFK5R, S4A, S4D, S4D,K5R versions of pVenus-N1-AurA and K391R, S390A, S390D versions of pVenus-C1-Nek2A were generated using standard mutagenesis techniques. Full cloning details available upon request.5.4.−1 tetracycline hydrochloride . Expression of Venus-tagged constructs was achieved by electroporation using Invitrogen Neon system according to the manufacturers' instructions. Cells were analysed 12–48 h following electroporation.U2OS cells  or U2OS tet-OFF cells expressing biotinylatable ubiquitin  were cull-cysteine (STLC) treatment for 16 h. Cells were collected by mitotic shake-off and forced into mitotic exit by silencing the SAC using Aurora B inhibitor ZM447439 for 70 min.Synchronization in mitotic exit was achieved using SAC-dependent mitotic arrest by kinesin-5 inhibitor S-trityl-5.5.8 cells were lysed in 10 ml lysis buffer  SDS, 1X protease inhibitor cocktail  and 50 mM N-ethylmaleimide in 1× PBS). The lysate was applied to 450 µl beads (Dynabeads His-Tag Isolation & Pulldown from Life Technologies) and incubated for 1 h at room temperature. The beads were then washed twice with wash buffer 1  followed by once with wash buffer 2 (wash buffer 1 + 10 mM imidazole). Three sequential elutions were performed by incubating the beads with 1 ml elution buffer  for 30 min at room temperature. The eluate was pooled together and concentrated using a Vivaspin Centrifugal Concentrator.For each sample, about 105.6.Samples were processed by the Cambridge Centre For Proteomics. MS/MS spectra were searched using the Mascot search engine (Matrix Science) against a Swissprot database restricted to human entries, with a maximum of three missed cleavages allowed. Carbamidomethylation was set as fixed modification while methionine oxidation and GlyGly modification on K were included as variable modifications. The search was performed with an initial mass tolerance of 1 Da for the precursor ion and 0.6 Da for the MS/MS spectra. Error-tolerant search was allowed to identify other potential modifications.5.7.6 cells were lysed in 100 µl lysis buffer  triton; 1×protease inhibitor cocktail (Roche); 1 mM NaF; 1 mM Na3VO4; 50 mM N-ethylmaleimide). Following dilution with 400 µl dilution buffer , the lysate was applied to 5 µl GFP-Trap_A beads and incubated for 2 h at room temperature. The beads were then washed with dilution buffer followed by thrice with 1 ml wash buffer 1 (8 M urea + 1% (v/v) SDS in PBS) and once with wash buffer 2 (1% (v/v) SDS in PBS). To minimize the non-specific binding and the binding of Aurora A interactors, each wash with wash buffer 1 was performed on rollers for 5 min. The beads were then boiled in 5 μl elution buffer  for 10 min at 95°C and the eluted sample was separated from the beads by centrifugation. Blots were probed with GFP antibody  and biotin antibody , for quantitative detection of unmodified and ubiquitinated AurA-Venus, respectively, using a LI-COR Odyssey machine.Biotin–ubiquitin cells were transfected with indicated AurA-Venus construct for 48 h, and synchronized to SAC-dependent mitotic arrest using STLC, collected by mitotic shake-off and forced into mitotic exit by silencing the SAC using Aurora B inhibitor ZM447439 for 70 min. For each sample, about 5 × 105.8.4 cm−2 onto eight-well plastic-bottom slides  for time-lapse analyses. Imaging medium was L-15 supplemented with FBS and antibiotics. Time-lapse imaging was carried out on an Olympus CellR imaging platform comprised of Olympus IX81 motorized inverted microscope, Orca CCD camera , motorized stage  and 37°C incubation chamber . Epifluorescent and DIC images were acquired with 1 × 1 bin using appropriate filter sets and 40× NA 1.3 oil objective, at 2 min intervals. Image sequences were exported as 12-bit tiff files for analysis in ImageJ. Degradation curves and rate curves were plotted with an in-house script using R environment, with degradation rates calculated from the gradient of the tangent at each point of degradation curves after smoothing [U2OS cells were electroporated with AurA-Venus or Venus-Nek2A constructs and seeded at 2 × 10moothing .


As a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products.Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates.The cellobiose and glucose inhibition of thermostable cellulases from GH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of product inhibition. Tricoderma reesei (Tr). The major component of the Tr cellulolytic system is the glycoside hydrolase (GH) family 7 , where vi and v0 are the rates measured in the presence and absence of inhibitor, respectively, and [I] is the concentration of inhibitor. To find the IC50, the data are first fitted to hyperbolae in the following form:A simplified approach for assessing the inhibitory strength is to measure the C1 and C2 found by the fitting are further used to calculate the IC50 value using Equation 2:In the fitting of the data, the substrate concentration ([S]) is fixed to the value used in the experiments. The above value of [S] and the values of empirical constants IC50 is an empirical parameter and its value may depend on the concentration of the substrate  used in the measurement of the IC50. If and how the IC50 value depends on [S]/KM depends on the type of inhibition. In the case of competitive inhibition, the relationship among IC50, Ki and [S]/KM is given as follows:The IC50 is well below its KM value, the resulting IC50 value is close to the true Ki value. However, if [S] is near saturating for the enzyme, the inhibition appears to be weak, as the resulting IC50 is much higher than Ki. The situation is opposite in the case of un-competitive inhibition, as in this case we have the following:Thus, if the inhibition is competitive and the [S] used in the measurement of the IC50, Ki (there are two different Kis now) and [S]/KM is more complicated, and whether the inhibition appears to be stronger at low or high [S]/KM ratio depends on which type of inhibition (competitive or un-competitive) is dominating. However, in the case of pure non-competitive inhibition, IC50 = Ki, so IC50 represents the value of the true Ki at any substrate concentration used for its measurement.In the case of mixed inhibition, the interplay among 14C-BC. Thermostable GH7 CBHs AtCel7, TaCel7A, and CtCel7A , where DCB and DCB=0 represent the degree of conversion of 14C-BC in the presence and absence of cellobiose, respectively  demonstrates that the inhibition of CBHs by the cellobiose released during hydrolysis was significant . The degree of conversion resulting from the activity of the EG was measured in a separate experiment . The resulting Ki values are listed in Table IC50 and Ki values of TrCel7A resulted in standard enthalpy changes of 63.6 ± 2.6 kJ mol-1 , and the inhibition of cellulose hydrolysis appears to be weak. This scenario has been proposed to explain the differences in the inhibitory strength of xylo-oligosaccharides toward CBHs acting on MUL and cellulose    is the IC50 for cellobiose previously determined) to Equation 5 to create Equation 7:The glucose inhibition of CBHs with  glucose ) and als] Figure D, the inGlc and DGlc=0 represent the degree of conversion of 14C-BC in the presence and absence of added glucose, respectively; [Glc] is the concentration of added glucose; [14CBC] is the 14C-BC concentration used in the experiment; and C1, C2 and H are empirical constants. The values of C1, C2 and H obtained by the fitting of the data to Equation 7 were used to calculate the IC50 for glucose according to Equation 6. The glucose inhibition of GH7 CBHs was more than two orders of magnitude weaker than cellobiose inhibition  over all time points was used in plotting (DCB/DCB=0) versus [cellobiose]  was from Fluka. D-[U-14C-BC was prepared by laboratory fermentation of the Gluconobacter xylinum strain ATCC 53582 [14C] glucose carbon source [14C-BC had a specific activity 450,000 DPM mg-1. 14C-BMCC was prepared by the limited acid hydrolysis of 14C-BC, and 14C-amorphous cellulose was prepared from 14C-BMCC by dissolution and regeneration from phosphoric acid [CC 53582  in the pn source . 14C-BC ric acid . The totTrCel7A was purified from the culture filtrate of Tr QM 9414 as described previously [AtCel7A, CtCel7A or TaCel7A were kindly provided by Terhi Puranen from Roal Oy . CBHs were heterologously expressed in a Tr strain lacking the genes of four major cellulases [TaCel7A was provided with the carbohydrate binding module of TrCel7A [AtCel7A and TaCel7A) or with 20 mM sodium phosphate, pH 6.5 (in the case of CtCel7A). CBHs were eluted with a linear gradient of 0 – 0.3 M NaCl in equilibration buffer.eviously . Culturellulases ,44. The  TrCel7A ,44. CBHsTrCel6A was purified from the culture filtrate of Tr QM 9414 as described previously [CtCel6A heterologously expressed in Tr originated from Roal OY  and was kindly provided by Matti Siika-Aho from VTT . CtCel6A was purified on a DEAE-Sepharose column after buffer exchange on a Toyopearl HW-40 column. For ion-exchange chromatography on DEAE-Sepharose, the column was equilibrated with 20 mM sodium phosphate (pH 7.0), and CtCel6A was eluted with a linear gradient of 0 – 0.5 M NaCl in equilibration buffer.eviously ,73. The TrCel7B, TrCel5A, and TrCel12A were purified from the culture filtrate of Tr QM 9414 as described previously [N188BG was purified from Novozyme®188 as described in [eviously ,74,75. Nribed in .280 values.The concentration of the enzymes was measured from the absorbance at 280 nm using theoretical ϵ14C-BC. For that, 14C-BC (0.25 g l-1) was incubated (without stirring) with a mixture of CBH (0.25 μM), TrCel5A (0.025 μM) and N188BG (0.06 μM) in 50 mM sodium acetate buffer pH 5.0 containing BSA (0.1 g l-1). At selected times, 0.2-ml aliquots were withdrawn and added to 20 μl 1 M NaOH to stop the reaction. Residual cellulose was separated by centrifugation , and radioactivity in the supernatant was quantified using liquid scintillation counting. The degree of cellulose degradation was calculated from the ratio of radioactivity in the supernatant to the total radioactivity in the hydrolysis mixture. In the case of inhibition studies, the reactions were supplied with cellobiose and glucose at different concentrations, and N188BG was omitted.The activity and inhibition of GH7 CBHs were assessed by following the synergistic hydrolysis of -1). Reactions were stopped by the addition of NH3 , and the released 4-methylumbelliferone was quantified by fluorescence using excitation and emission wavelengths of 360 nm and 450 nm, respectively.For the inhibition of enzyme acting on the low-Mw substrate, the initial rates of the hydrolysis of MUL in the presence and absence of added cellobiose were followed. MUL (5 μM) was incubated with CBH (10 nM) in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l14C-BMCC. 14C-BMCC (0.25 g l-1) was incubated (with shaking at 350 rpm) with CBH (0.25 μM) and N188BG (0.06 μM) in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l-1). The remainder of the procedure was identical to that described for GH7 CBHs. In the case of inhibition studies, the reactions were supplied with cellobiose and glucose at different concentrations, and N188BG was omitted.GH6 CBHs were assessed by observing the hydrolysis of 14C-BC was performed identically to the procedure described for GH7 CBHs, but the CBH component was 0.25 μM TrCel6A.The cellobiose inhibition of the synergistic hydrolysis of 14C-amorphous cellulose. 14C-amorphous cellulose (0.5 g l-1) was incubated (with shaking at 700 rpm) with EG in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l-1) in the presence and absence of added cellobiose. The concentration of EG was 2.5 nM, 5.0 nM, and 50 nM for TrCel7B, TrCel5A, and TrCel12A, respectively. The remainder of the procedure was identical to that described for GH7 CBHs.EGs were assessed on Acremonium thermophilum; BG: β-glucosidase; BSA: Bovine serum albumin; CB: Cellobiose; 14C-BC: 14C-labeled bacterial cellulose; CBH: Cellobiohydrolase; 14C-BMCC: 14C-labeled bacterial microcrystalline cellulose; Ct: Chaetomium thermophilum; Di: Degree of conversion in the presence of inhibitor i; D0: Degree of conversion in the absence of inhibitor; DP: Degree of polymerization; EG: Endoglucanase; GH: Glycoside hydrolase; Glc: Glucose; MUL: 4-methylumbelliferyl-β-lactoside; pNP: Para-nitrophenol; pNPL: Para-nitrophenyl-β-lactoside; Ta: Thermoascus aurantiacus; Tr: Trichoderma reeseiAt: The authors declare that they have no competing interests.HT and PV designed and performed the experiments. PV wrote the paper. Both authors read and approved the final manuscript.14C-BC by EG, TrCel5A. Figure S5. Inhibition of EGs, TrCel5A and TrCel12A, by cellobiose. Inhibition of GH7 CBHs by cellobiose at 50°C. Figure S2. Inhibition of GH7 CBHs by cellobiose at 25°C. Figure S3. Analysis of the inhibition of GH7 CBHs by cellobiose at 25°C and 50°C. Figure S4. Hydrolysis of Click here for file


Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis.Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell.Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the transcriptional activity of AIB1. The overexpression of AIB1, a transcriptional coactivator, promotes pre-neoplastic changes and cancer initiation in animal models Most of the studies related to AIB1 and cell-cycle regulation have focused on G1/S progression. During this phase, AIB1 is localized, along with ERα, to the active promoter of cyclin D1 The events of mitosis, including reorganization of the cellular architecture and chromosome condensation, require fine tuning to ensure the precision of the cell-cycle. These processes are coordinated by different families of kinases that trigger protein phosphorylation cascades. The cyclin-dependent kinase Cdk1 is a key regulator of the onset of mitosis. Activation of Cdk1 during late G2 initiates the cellular reorganization which also involves the activity of three other kinase families: Aurora, Polo-like (Plk) and NIMA-related (Nrk) kinases. Although both Aurora A and Plk1 are activated during early G2 With the present study, we demonstrate that AIB1 associates with Cdk1 and undergoes phosphorylation just at the entry to mitosis. This phosphorylation event is dependent on cyclin B but not cyclin A, further supporting the phosphorylation of AIB1 at the onset of mitosis. Interestingly, neither ubiquitylation nor protein degradation were associated with this phosphorylation. AIB1 remains phosphorylated throughout M phase and is dephosphorylated only at the exit from mitosis, coinciding with activation of PP1 and the inactivation of Cdk1. Importantly, phosphorylation at the defined sites did not alter the transcriptional activity of AIB1 but rather excludes this factor from chromatin during mitosis, perhaps reflecting a mechanism to prevent abnormal transcription during M phase. Collectively, these results reveal a novel mechanism for regulating AIB1 during mitosis by which chromatin exclusion and subcellular redistribution of AIB1 during mitosis may restrict its transcriptional activity.Regulation of AIB1 activity and stability is mediated by several post-translational modifications. To study the cell cycle-dependent post-translational modifications of AIB1, we analyzed synchronized cultures of HeLa cells. These cells were arrested at different stages of the cell cycle  or mitotic (M) culture lysates revealed the presence of Cdk1 in AIB1 immunocomplexes, suggesting a functional relationship in vivo between these proteins  is responsible for the majority of this dephosphorylation activity To further substantiate the dephosphorylation of AIB1 at the exit from mitosis, rounded up cells were separated from adherent cells 3 hours after their release from nocodazole-mediated arrest and were analyzed by western blotting. Interestingly, AIB1 was completely dephosphorylated in adherent cells that represent G1, whereas the rounded up fraction of cells retained at M contained only phosphorylated AIB1 as evidenced by the slower mobility of this band .de novo protein synthesis. Based on flow cytometry analysis, cells treated with cycloheximide were able to complete mitosis, although no cyclin D1 was synthesized  or both PP1 and PP2A (0.5 µM). Okadaic acid blocked the dephosphorylation of AIB1 in HeLa cells in a dose-dependent manner .Calyculin A is a more effective inhibitor of both PP1 and PP2A than okadaic acid. Hence, we then compared these inhibitors in nocodazole-arrested HeLa cells. The dephosphorylation of AIB1 was only partially inhibited in the presence of okadaic acid . Treatmein vitro kinase assays with complexes of cyclin B1/Cdk1 (To identify the site(s) targeted by Cdk1, we generated various AIB1 fragments (named A through E) that span the full-length sequence . Fragmen B1/Cdk1  or cycli B1/Cdk1 . Fragmen B1/Cdk1 , furthervs. C S728A). We also generated other point mutations and found that S867A combined with S728A completely abolished the Cdk1-mediated phosphorylation , constituting a bipartite recognition motif for cyclin-dependent kinases rylation , suggestWe next investigated whether the phosphorylation state of AIB1 might modulate its sub-cellular localization. Proliferating MCF-7 cells were stained with anti-AIB1 antibodies and mitotic cells were identified by DAPI or anti-tubulin staining. Our results reveal that AIB1 is excluded from chromatin in mitotic cells , suggestTo assess the effects of phosphorylation on the transcriptional activity of AIB1, we fused full-length AIB1 to the DNA binding domain of Gal4  and co-transfected it with the luciferase reporter driven by the Gal4 responsive element. COS1 cells were treated with nocodazole prior to analysis. No significant differences were detected between AIB1 with the mutation at S728 and wild-type AIB1 . Alternain vitro, suggesting that this phosphorylation event does not occur at G2 but rather exclusively at M phase, when the cyclin B/Cdk1 complex is active in dividing cells.Phosphorylation of proteins during mitosis and their dephosphorylation at the exit from mitosis require precise regulation to maintain the integrity of the cell-cycle. The function of the transcriptional coactivator AIB1 has been studied extensively in the progression of G1 to S but little is known about its role in M phase. AIB1 is the target of various kinases that regulate both its function and its turnover. Our results demonstrate that AIB1 also serves as a substrate of Cdk1/cyclin B just at the entrance of mitosis, thus providing a new mechanism for regulating AIB1 during the cell cycle. We found that phosphorylation of AIB1 occurs solely in mitotic cells and that this molecule remains phosphorylated throughout mitosis. Consistent with this, Cdk1 associated with cyclin B not cyclin A was able to phosphorylate AIB1 Mitogenic stimuli that promote cell-cycle progression through G1/S trigger the activation of signaling kinases such as ERKs and AKT. Phosphorylation of AIB1 by ERKs at multiple residues results in the recruitment of p300 and enhances its coactivation capacity in vitro kinase assay. Ser728 is potently phosphorylated by Cdk1 and is within the motif (S/T)PX(R/K) (where X is any amino acid) found in most Cdk substrates. Both sites have recently been reported to be phosphorylated using a high throughput proteomics analysis We have identified two serines (728 and 867) which were phosphorylated by Cdk1 in an During interphase, transcription factors and related proteins are frequently redistributed within the nucleus of cells. Many of these proteins (such as RNA polymerase II) are displaced from chromatin during mitosis, whereas others like TFIID and TFIIB can remain associated with active gene promoters during mitosis HeLa, MCF-7 and COS-1 cells (ATCC) were maintained in DMEM – 10% FBS (Invitrogen). Sf9 cells (Invitrogen) were cultured in SF-900 II medium (Invitrogen). Transfections in COS-1 cells were performed with FuGene 6 (Roche) according to manufacturer instructions. For synchronization, cells were deprived of serum for 24 h and released in the presence of one of the following inhibitors: 16 h, 5 µM cyclosporin to arrest at G0/G1; 24 h with 100 nM wortmannin for late G1, 24 h with 400 µM mimosine for G1/S, 48 h with 5 mM hydroxyurea for S-phase, 24 h with 10 µM etoposide for G2/M and for 17 h with 150 ng/ml nocodazole for M-phase (prometaphase) followed by shake-off to collect mitotic cells. All inhibitors were purchased from Sigma. Mitotic cells were washed and released for the indicated times in the presence or absence of 10 µg/ml cycloheximide (Sigma), 20 µM MG132 , 0.1–1 µM okadaic acid  and 20 nM calyculin A . Alternatively, mitotic cells were resuspended in nocodazole containing media supplemented with 20 µM PD98059 (Sigma) or 10 µM purvalanol A  alone and/or okadaic acid or calyculin A for 2 h.Cells were harvested by trypsin treatment, washed with PBS and resuspended in 70% ethanol to permit fixation and permeabilization at −20°C. Propidium iodide-stained cells  were analyzed using a flow cytometer .2HPO4/NaH2PO4 and 0.1 M Tris-HCl pH 8, sonicated for 30 s and centrifuged for 15 min at 16000×g. Cleared supernatant was diluted 1∶1 in PBS, and His6-tagged proteins were purified by Ni2+-NTA affinity chromatography (Qiagen) in the presence of 10 mM Imidazole (Merck), 1 mM AEBSF (Roche), 20 µM MG132, 10 mM N-etilmaleimide (Sigma) and 5 nM ubiquitin aldehyde (Santa Cruz Biotech) during 2 h at room temperature. Agarose beads were then washed once with buffer A (same as lysis buffer but with 10 mM imidazol), once with buffer B , four times with decreasing concentrations of Triton X-100 (starting at 0.2%) in buffer C . Finally, ubiquitylated proteins were eluted by boiling in Laemmli buffer.Cells were washed with ice-cold PBS and lysed with commercial lysis buffer  supplemented with protease cocktail inhibitors (Roche), 1 mmol/l NaF and up to 300 mM NaCl. His6-tagged proteins were lysed with 6 M guanidium-HCl, 0.1 M NaSubcellular fractionation was done as previously reported Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 ; monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents). Phospho-specific anti-pAIB1(S728) were raised in rabbits against the peptide KQEQL(pS)PKKKENNA. Sera from the immunized rabbits were cleared with the unphosphorylated peptide.Pre-cleared whole-cell lysates (1 mg) were immunoprecipitated with polyclonal AIB1 antibodies (generated in our laboratory) during 4 h at 4°C. Protein A-agarose was added to immunocomplexes and incubated for 2 more hours. Immunocomplexes were washed three times with lysis buffer and solubilized by boiling in Laemmli buffer. Half of the immunoprecipitation was analyzed by western blot. Preimmune serum was used as negative control.Plasmid constructs pGEX4T-1-AIB1(1–555), pGEX4T-3-AIB1(693–933), pGEX4T-3-AIB1(934–1031) and pGEX4T-1-AIB1(1032–1424) were generated by subcloning cleaved fragments from pcDNA3.1+AIB1 with SspI and EcoRI. Construct pGEX4T-1-AIB1(556–693) was generated by PCR to add EcoRI-SalI sites. Point mutations S728A, S728E, S860A, S867A and R739A/L741A were generated using QuickChange site-directed mutagenesis kit (Stratagene). Mutations were verified by sequencing.Kinase assay- Recombinant full-length AIB1 protein produced in Sf9 cells (1 µg) or GST-AIB1 fragments attached to the sepharose beads (15 µl) were subjected to in vitro phosphorylation in a final volumen of 30 µl with kinase buffer, 10 units of Cdk1-cyclin B1 complex , 200 µM ATP and 5 µCi [γ-32P]-ATP (Perkin Elmer). Reactions were incubated for 1 h at 30°C, analyzed by SDS-PAGE and exposed for autoradiography. 1 µg of Histone H1 (Sigma) and 1 µg of GST-Retinoblastoma (aminoacids 792–928) were used as positive controls. In vitro phosphorylation by Cdk1-cyclin A2 complex  was performed similarly as above, except that 100 ng of Cdk1-cyclin A2 complex were used per reaction.