Activity Based Protein Profiling

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim -glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-gl...

The fusion of enzymatic proteins represents a dynamic frontier in biotechnology and enzymatic engineering. This in-depth review looks at the many different ways that fusion proteins can be used, showing their importance in biosensing, gene therapy, targeted drug delivery, and biocatalysis. Fusion proteins have shown an astounding ability to improve and fine-tune biological functions by combining the most beneficial parts of different enzymes. Our first step is to explain how protein fusion increases biological functions. This will provide a broad picture of how this phenomenon has changed many fields. We dissect the intricate mechanisms through which fusion proteins orchestrate cellular processes, underscoring their potential to revolutionize the landscape of molecular biology. We also explore the complicated world of structural analysis and design strategies, stressing the importance of molecular insights for making the fusion protein approach work better. These insights broaden understanding of the underlying principles and illuminate the path toward unlocking untapped potential. The review also introduces cutting-edge techniques for constructing fusion protein libraries, such as DNA shuffling and phage display. These new methods allow scientists to build a molecular orchestra with an unprecedented level of accuracy, and thus use fusion proteins to their full potential in various situations. In conclusion, we provide a glimpse into the current challenges and prospects in fusion protein research, shedding light on recent advancements that promise to reshape the future of biotechnology. As we make this interesting journey through the field of enzymatic protein combination, it becomes clear that the fusion paradigm is about to start a new era of innovation that will push the limits of what is possible in biology and molecular engineering.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomassdegrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-Larabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-Larabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-Larabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig− Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-Larabinofuranosidases.

The use of triethylsilane as a carbocation scavenger in the presence of trifluoroacetic acid in dichloromethane leads to increased yields, decreased reaction times, simple work-up and improved selectivity for the deprotection of t-butyl ester and t-butoxycarbonyl sites in protected amino-acids and peptides in the presence of other acid-sensitive protecting groups such as the benzyioxycarbonyl, 9-fluorenylmethoxycarbonyl, O-and S-benzyl and t-butylthio groups.

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore-quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala(4)]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala(8)]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala(5)]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control,...

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala(4)]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala(8)]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala(5)]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control,...

Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.

Formylation of amino groups is a critical reaction involved in several biological processes including post-translational modification of histones. The addition of a formyl group (CHO) to the N-terminal end of a peptide chain generates biologically active molecules. N-formyl-peptides can be produced by different methods. We performed the N-formylation of two chemotactic hexapetides, Met1-Leu2-Lys3-Leu4-Ile5-Val6 and Met1-Met2-Tyr3-Ala4-Leu5-Phe6, carrying out the reaction directly on peptidyl-resin following pre-activation of formic acid with N,N-dicyclohexylcarbodiimmide (DCC) in liquid phase. The overnight incubation at 4˝C resulted in a significant increase in production yields of formylated peptides compared to the reaction performed at room temperature. The method is consistently effective, rapid, and inexpensive. Moreover, the synthetic strategy can be applied for the formylation of all primary amines at N-terminus of peptide chains or amino groups of lysine side-chains in solid phase.

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala(4)]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala(8)]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala(5)]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control,...

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala(4)]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala(8)]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala(5)]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control,...

The development and application of bioorthogonal two-step labeling techniques receives much attention. Employing bifunctional proteasome probe 2 the efficiency of two-step labeling of recently published biotinylated cyclooctynes 3-5 is compared to Staudinger-Bertozzi ligation in cell extracts and living cells. While cyclooctynes 3-5 react faster and at a much lower concentration then the Staudinger-Bertozzi benchmark, background labeling is considerable with these reagents.

SUMO is ap ost-translational modifier critical for cell cycle progression and genome stability that playsarole in tumorigenesis,t hus rendering SUMO-specific enzymes potential pharmacological targets.H owever,t he systematic generation of tools for the activity profiling of SUMO-specific enzymes has proven challenging.W edeveloped adiversifiable synthetic platform for SUMO-based probes by using ad irect linear synthesis method, which permits N-and C-terminal labelling to incorporate dyes and reactive warheads,r espectively.I nt his manner,a ctivity-based probes (ABPs) for SUMO-1, SUMO-2, and SUMO-3-specific proteases were generated and validated in cells using gel-based assays and confocal microscopy. We further expanded our toolbox with the synthesis of aK 11-linked diSUMO-2 probe to study the proteolytic cleavage of SUMO chains.T ogether,t hese ABPs demonstrate the versatility and specificity of our synthetic SUMO platform for in vitro and in vivo characterization of the SUMO protease family.

Combination regimens that include artemisinin derivatives are recommended as first line antimalarials in most countries where malaria is endemic. However, the mechanism of action of artemisinin is not fully understood and the usefulness of this drug class is threatened by reports of decreased parasite sensitivity. We treated Plasmodium falciparum for periods of a few hours to mimic clinical exposure to the short half-life artemisinins. We found that drug treatment retards parasite growth and inhibits uptake of hemoglobin, even at sublethal concentrations. We show that potent artemisinin activity is dependent on hemoglobin digestion by the parasite. Inhibition of hemoglobinase activity with cysteine protease inhibitors, knockout of the cysteine protease falcipain-2 by gene deletion, or direct deprivation of host cell lysate, significantly decreases artemisinin sensitivity. Hemoglobin digestion is also required for artemisinin-induced exacerbation of oxidative stress in the parasite c...

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to , the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces Here, we explore the possibility that the limited colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on infection. The P69 family members P69C and P69F, and an unannotated lipase (So...

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim -glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-gl...

Introduction Panepophenanthrin (1) is a natural product that inhibits ubiquitin-activating enzyme (E1) and was isolated by Sekizawa and co-workers in 2002 from the mushroom strain Panus rudis Fr. IFO8994. [1] As ubiquitin-activating enzyme (E1) plays an important role in the ubiquitin-proteasome pathway (UPP), which regulates a variety of important cellular processes by degradation or processing of target proteins, an inhibitor of ubiquitin-activating enzyme (E1) would be a promising drug candidate for cancers, inflammation, and neurodegenerative disease.

We employed a comprehensive approach of target-based virtual high-throughput screening to find potential hits from the ZINC database of natural compounds against cysteine proteases falcipain-2 and falcipain-3 (FP2 and FP3). Molecular docking studies showed the initial hits showing high binding affinity and specificity toward FP2 were selected. Furthermore, the enzyme inhibition and surface plasmon resonance assays were performed which resulted in a compound ZINC12900664 (ST72) with potent inhibitory effects on purified FP2. ST72 exhibited strong growth inhibition of chloroquine-sensitive (3D7; EC 50 = 2.8 µM) and chloroquine-resistant (RKL-9; EC 50 = 6.7 µM) strains of Plasmodium falciparum. Stage-specific inhibition assays revealed a delayed and growth defect during parasite growth and development in parasites treated with ST72. Furthermore, ST72 significantly reduced parasite load and increased host survival in a murine model infected with Plasmodium berghei ANKA. No Evans blue staining in ST72 treatment indicated that ST72 mediated protection of blood-brain barrier integrity in mice infected with P. berghei. ST72 did not show any significant hemolysis or cytotoxicity against human HepG2 cells suggesting a good safety profile. Importantly, ST72 with CQ resulted in improved growth inhibitory activity than individual drugs in both in vitro and in vivo studies.

Synthetic vaccines, based on antigenic peptides that comprise MHCÀ I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Tolllike receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam 3 CysSK 4. A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block preprepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.

Synthetic vaccines, based on antigenic peptides that comprise MHCÀ I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Tolllike receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam 3 CysSK 4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block preprepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.

Strains of Clostridium difficile produce a number of surface-localized proteins, including the Slayer proteins (SLPs) and other proteins that have suspected roles in pathogenesis. During the Third International C. difficile Symposium (Bled, Slovenia, September 2010) discussions were held on standardization of nomenclature. Gene designations were proposed for the large family of cell wall proteins that are paralogues of the SLP and contain putative cell wall binding motifs. This paper summarizes the agreed nomenclature, which we hope will be used by research groups currently active in the field.

ALDH3H1 is partially inhibited by Alda-1 and DDZ. Enzymatic activities of the treated enzymes were measured spectrophotometrically using aliphatic aldehydes; hexanal (2mM), nonanal (0.5mM), trans-2-decenal (0.25mM) and trans-2-dodecenal (0.25mM). Error bars represent SEM of n=3.

Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme®). The structures of IDUA when complexed with the inhibitors in a non-covalent transition state mimicking form and a covalent enzyme-bound form provide insights into its conformational itinerary. Inhibitors 1–3 adopt a half-chair conformation in solution (4H3 and 3H4), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1–3 may need to overcome an energy barrier in order to switch from the 4H3 conformation to the transition state (2, 5B) binding conformation before reacting and adopting a covalent 5S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.

Plasmodium falciparum, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of P. falciparum overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present ...

Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector control have reduced the global malaria burden in the past decade; however, the emerging threat of drug resistance and suboptimal vaccine efficacies threaten global eradication efforts. Unlocking novel drug and vaccine targets while simultaneously mitigating spread of resistant strains seems to be the need of the hour. Protein-protein interactions (PPIs), an integral part of hostpathogen cross-talk and parasite survival, have only recently emerged as promising drug targets. Large PPI networks (interactome) are being developed to better our understanding of various parasite biochemical pathways. In this chapter, we throw light on several newly characterized protein-protein interactions between the host (humans) and parasite (plasmodium) in key processes such as hemoglobin degradation, enzyme regulation, protein export, egress, invasion, and drug resistance and further discuss their viability for development as novel chemotherapeutic targets.

Descrevemos uma síntese do esqueleto carbônico dos sesquiterpenos do grupo eudesmano substituído nos carbonos 1 e 6, que se baseia na versão multicomponente da reação de Diels-Alder. We report a synthesis of the 1,6-disubstituted eudesmane sesquiterpene carbon skeleton, based upon a multicomponent Diels-Alder reaction.

Descrevemos uma síntese do esqueleto carbônico dos sesquiterpenos do grupo eudesmano substituído nos carbonos 1 e 6, que se baseia na versão multicomponente da reação de Diels-Alder. We report a synthesis of the 1,6-disubstituted eudesmane sesquiterpene carbon skeleton, based upon a multicomponent Diels-Alder reaction.

Two closely related phenyl selenyl based boron-dipyrromethene (BODIPY) turn-on fluorescent probes for the detection of hypochlorous acid (HOCl) were synthesized for studies in chemical biology; emission intensity is modulated by a photoinduced electron-transfer (PET) process. Probe 2 intrinsically shows a negligible background signal ; however, after reaction with HOCl, chemical oxidation of selenium forecloses the PET process, which evokes a significant increase in fluorescence intensity. The fluorescence intensity of probes 1 and 2 with HOCl involves an ~18 and ~50-fold enhancement compared with the respective responses from other reactive oxygen/nitrogen species (ROS/RNS) and low detection limits (30.9 nm for 1 and 4.5 nm for 2). Both probes show a very fast response with HOCl; emission intensity reached a maximum within 1 s. These probes show high selectivity for HOCl, as confirmed by confocal microscopy imaging when testing with RAW264.7 and MCF-7 cells.

The development and application of bioorthogonal two-step labeling techniques receives much attention. Employing bifunctional proteasome probe 2 the efficiency of two-step labeling of recently published biotinylated cyclooctynes 3-5 is compared to ...

Depsidomycin is a cyclic heptadepsi-peptide isolated from the cultured broth of Streptomyces lavendofoliae MI951-62F2. It exhibits significant antimicrobial and immunosuppressive activity. The total synthesis of a depsidomycin analogue in which 1,2-piperazine-3-carboxylic acid was substituted with proline is described. After several trials using different strategies, the desired depsidomycin analogue was obtained via stepwise synthesis starting by the amino acid 'head' and macrolactonization under Yamaguchi conditions. The cyclic depsipeptide was evaluated to have an minimum inhibitory concentration (MIC) of 4 µg/ml against H37RV and 16 µg/ml against MDR clinical strains of MTB (MDR-MTB), while the linear precursor 8 also had MICs of 4 and 16 µg/ml for the susceptible and resistant strains, respectively.

Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.

The development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosic bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mecha...

Protein redox chemistry constitutes a major void in knowledge pertaining to photoautotrophic system regulation and signaling processes. We have employed a chemical biology approach to analyze redox sensitive proteins in live Synechococcus sp. PCC 7002 cells in both light and dark periods, and to understand how cellular redox balance is disrupted during nutrient perturbation. The present work identified 300 putative redox-sensitive proteins that are involved in the generation of reductant, macromolecule synthesis, and carbon flux through central metabolic pathways, and may be involved in cell signaling and response mechanisms. Furthermore, our research suggests that dynamic redox changes in response to specific nutrient limitations, including carbon and nitrogen limitations, contribute to the regulatory changes driven by a shift from light to dark. Taken together, these results contribute to a high-level understanding of post-translational mechanisms regulating flux distributions and suggest potential metabolic engineering targets for redirecting carbon toward biofuel precursors.

Small bis-conjugates helix A329–350-PEG-triazole-PEG-helix B508–526 (41 residues), prepared through click chemistry of PEGylated peptide derivatives, bind to CaM with nanomolar affinity, behaving as mimics of the Kv7.2 native fragment (239 residues).

Metalloproteases (ADAMs, MMPs) are multidomain proteins that play key roles in extracellular matrix remodelling and degradation, in cell-cell and cell-matrix interactions and in the proteolytic liberation of membrane-anchored proforms of cytokines and growth factors, the so-called ectodomain shedding. In this work we describe the development of photoactivatable activity-based probes with which active [a]

With nearly 140 a-glycosidases in 14 different families, plants are well equipped with enzymes that can break the a-glucosidic
bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of a-glycosidases in plants
using a-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana),
these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident
a-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident a-mannosidase-II Hybrid
Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular
a-glycosidases such as a-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and
a-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of a-glycosidases
generates pH-dependent signals that can be suppressed by a-glycosidase inhibitors in a broad range of plant species. To
demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the
production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active
glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase
activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes
for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active a-glycosidases and
other hydrolases can be analyzed through ABPP in model and nonmodel plants.

Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10 6 M -1 s -1 . The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CHdCH-CON(CH 2 -1-Naphth) 2 is the most potent compound and it inhibits caspase-3 with a k 2 value of 5620000 M -1 s -1 . The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1′ moiety.

Background: Diabetes is a chronic endocrine disorder caused by lack of insulin and/or reduced insulin activity leads hyperglycemia and abnormalities in carbohydrate, fat-protein metabolism. Numerous medicinal plants have been used for the management of diabetes mellitus in various traditional systems of medicine worldwide as they are a great source of biological constituents and many of them are known to be effective against diabetes due to their antihyperglycemic activities. Methods: This current research work was focused on antihyperglycemic proteins profiling from seeds and leaves of aqueous and methanolic extracts of Momordica charantia, Trigonella foenum-graneum, Triticum aestivum, Solanum tuberosum and Nicotiana tobaccum. Present study was undertaken to analyze the protein profiles from aqueous and methanolic extracts of seeds and leaves of some medicinally important test plants. Crude protein, Osmotin I and II and Polypeptide-P proteins from seeds and leaf. Result: The study indicated the presence of antihyperglycemic proteins, thus supporting its traditional medicinal practices. SDS-PAGE revealed that the band of osmotin I at 26KDa. Osmotin I and osmotin II was recovered from seeds and leaves of aqueous and methanolic fraction of wheat, tobacco and potato. The band of Polypeptide-P was obtained at 11KDa from the aqueous and methanolic extracts of seed as well as leaves of bittermelon and fenugreek. Conclusion: The methanolic extract of test plants seeds and leaves contained antihyperglycemic proteins except polypeptide-p in aqueous fraction. These proteins act as artificial insulin or analogue for insulin/ animal protein adeponectin or thaumatin. These proteins should be subjected to further purification so as to explore the analysis of the mechanism of action under in vivo system i.e.; in animal cell lines to observe the bio efficacy of these components in animal model system. Therefore, it may further be serves as an alternative drug of choice.

The inside cover picture shows the plant-pathogen-derived natural product Syringolin A, which was turned into an activity-based probe by attaching a fluorescent rhodamine moiety. This probe was then used to shed light on the labelling specificity of Syringolin A, demonstrating a highly specific binding to the eukaryotic 20S proteasome.

Dedicated to Professor Edwin Vedejs on the occasion of his 70th birthday tinct pattern of influence of each of the three compounds on cell division, differentiation, and apoptosis in HL-60 cells, thus suggesting that these effects are independent activities of the natural product. Scheme 4. Synthesis of lactone 8. DHP = 3,4-dihydro-2H-pyran, DMAP = 4-dimethylaminopyridine, HMDS = hexamethyldisilazide, PCC = pyridinium chlorochromate, PPTS = pyridinium para-toluenesulfonate, THP = tetrahydropyran-2-yl, TMS = trimethylsilyl. Scheme 5. Synthesis of the benzothiazolyl sulfones. Ts = tosyl. www.chemeurj.org Scheme 7. Functionalization of the spiroketal 7. DMSO = dimethyl sulfoxide, TBAF = tetra-n-butylammonium fluoride, Bn = benzyl. Scheme 8. Synthesis of the C19-C40 fragments. CBS = Corey-Bakshi-Shibata catalyst. Scheme 12. Synthesis of chiral homoallylic alcohol intermediate 41. CSA = camphorsulfonic acid, LDA = lithium diisopropylamide, PMB = para-methoxybenzyl, NMO = N-methylmorpholine-N-oxide. Scheme 14. Completion of the C1-C13 fragment. EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Scheme 13. Synthesis of key intermediate 4. DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, TES = triethylsilyl.