The oncofetal RNA-binding protein IGF2BP1 is a druggable, post-transcriptional super-enhancer of E2F-driven gene expression in cancer.

The IGF2 mRNA-binding protein 1 (IGF2BP1) is a non-catalytic post-transcriptional enhancer of tumor growth upregulated and associated with adverse prognosis in solid cancers. However, conserved effector pathway(s) and the feasibility of targeting IGF2BP1 in cancer remained elusive. We reveal that IGF2BP1 is a post-transcriptional enhancer of the E2F-driven hallmark in solid cancers. IGF2BP1 promotes G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators of this checkpoint like E2F1. This IGF2BP1-driven shortening of the G1 cell cycle phase relies on 3'UTR-, miRNA- and m6A-dependent regulation and suggests enhancement of cell cycle progression by m6A-modifications across cancers. In addition to E2F transcription factors, IGF2BP1 also stabilizes E2F-driven transcripts directly indicating post-transcriptional 'super'-enhancer role of the protein in E2F-driven gene expression in cancer. The small molecule BTYNB disrupts this enhancer function by impairing IGF2BP1-RNA association. Consistently, BTYNB interferes with E2F-driven gene expression and tumor growth in experimental mouse tumor models.

Posttranslational Chemical Mutagenesis: To Reveal the Role of Noncatalytic Cysteine Residues in Pathogenic Bacterial Phosphatases.

The field of chemical site-selective modification of proteins has progressed extensively in recent decades to enable protein functionalization for imaging, drug delivery, and functional studies. In this Perspective, we provide detailed insight into an alternative use of site-selective protein chemistry to probe the role(s) of unpaired Cys residues in the structure and function of disease relevant proteins. Phosphatases are important players in the successful infection of pathogenic bacteria, which represent a significant health burden, particularly in multi-drug-resistant strains. Therefore, a strategy for readily probing the key amino acid role(s) in structure and function may facilitate the targeting and inhibition of these virulence factors. With a dehydroalanine-based posttranslational chemical mutagenesis approach, it is possible to reveal hitherto unknown function(s) of noncatalytic Cys residues and confirm their role and interplay in pathogenic bacterial phosphatases. By selectively modifying reactive sulfhydryl side chains in different protein local environments, this posttranslational site-selective chemical mutagenesis approach reveals structural information about binding pockets and regulatory roles of the modified residues, which can be further validated by conventional site-directed mutagenesis. Ultimately, these new binding pockets can serve as templates for enhanced structure-based drug design platforms and aid the development of potent and specific inhibitors.

Chemo- and Regioselective Lysine Modification on Native Proteins.

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest p Ka was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein-antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

Synthetic compounds from an in house library as inhibitors of falcipain-2 from Plasmodium falciparum.

Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum. Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic organic compounds, including (E)-chalcones, (E)-N'-benzylidene-benzohydrazides and alkyl-esters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC50 values (8.5 ± 0.8 µM, 9.5 ± 0.2 µM and 4.9 ± 1.3 µM, respectively). The best inhibitor (compound 66) demonstrated non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy assays, we suggest a potential allosteric site for the interaction of this compound, located between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural biology tools and mass spectrometry to characterize the inhibition mechanisms of novel compounds targeting FP-2.

Nitric oxide inhibits the ATPase activity of the chaperone-like AAA+ ATPase CDC48, a target for S-nitrosylation in cryptogein signalling in tobacco cells.

NO has important physiological functions in plants, including the adaptative response to pathogen attack. We previously demonstrated that cryptogein, an elicitor of defence reaction produced by the oomycete Phytophthora cryptogea, triggers NO synthesis in tobacco. To decipher the role of NO in tobacco cells elicited by cryptogein, in the present study we performed a proteomic approach in order to identify proteins undergoing S-nitrosylation. We provided evidence that cryptogein induced the S-nitrosylation of several proteins and identified 11 candidates, including CDC48 (cell division cycle 48), a member of the AAA+ ATPase (ATPase associated with various cellular activities) family. In vitro, NtCDC48 (Nicotiana tabacum CDC48) was shown to be poly-S-nitrosylated by NO donors and we could identify Cys(110), Cys(526) and Cys(664) as a targets for S-nitrosylation. Cys(526) is located in the Walker A motif of the D2 domain, that is involved in ATP binding and was previously reported to be regulated by oxidative modification in Drosophila. We investigated the consequence of NtCDC48 S-nitrosylation and found that NO abolished NtCDC48 ATPase activity and induced slight conformation changes in the vicinity of Cys(526). Similarly, substitution of Cys(526) by an alanine residue had an impact on NtCDC48 activity. More generally, the present study identified CDC48 as a new candidate for S-nitrosylation in plants facing biotic stress and further supports the importance of Cys(526) in the regulation of CDC48 by oxidative/nitrosative agents.

RNA-binding proteins in cancer drug discovery.

RNA-binding proteins (RBPs) are crucial players in tumorigenesis and, hence, promising targets in cancer drug discovery. However, they are largely regarded as 'undruggable', because of the often noncatalytic and complex interactions between protein and RNA, which limit the discovery of specific inhibitors. Nonetheless, over the past 10 years, drug discovery efforts have uncovered RBP inhibitors with clinical relevance, highlighting the disruption of RNA-protein networks as a promising avenue for cancer therapeutics. In this review, we discuss the role of structurally distinct RBPs in cancer, and the mechanisms of RBP-directed small-molecule inhibitors (SMOIs) focusing on drug-protein interactions, binding surfaces, potency, and translational potential. Additionally, we underline the limitations of RBP-targeting drug discovery assays and comment on future trends in the field.

Structural stability of Staphylococcus xylosus lipase is modulated by Zn(2+) ions.

Lipases are well-known enzymes extensively used in industrial biotransformation processes. Besides, their structural and catalytic characteristics have attracted increasing attention of several industries in the last years. In this work, we used biophysical and molecular modeling tools to assess structural properties of Staphylococcus xylosus lipase (SXL). We studied the thermal unfolding of this protein and its zinc-dependent thermotolerance. We demonstrated that SXL is able to be active and stable at moderate temperatures, but this feature is only acquired in the presence of Zn(2+). Such characteristic indicates SXL as a zinc-dependent metallolipase.

Unveiling Druggable Pockets by Site-Specific Protein Modification: Beyond Antibody-Drug Conjugates.

Site-specific modification approaches have been extensively employed in the development of protein-based technologies. In this field, stability and activity integrity are the envisioned features of chemically modified proteins. These methods are especially used in the design of antibody-drug conjugates (ADCs). Nevertheless, a biochemical feature of the target protein in these reactions is often overlooked, residue specificity. Usually, in the course of developing chemical probes to modify a protein of interest (POI), specific amino acids are selected due to their reactivity. It is not critical which residue is modified as long as its modification does not compromise the POI's activity. However, no attention is paid as to why certain residues are preferentially modified over others. Physicochemical and structural constraints are often involved in the reactivity of the residue and account for the preferential modification. We propose that site-specific protein modification approaches can be applied beyond the development of ADCs or protein-drug conjugates, and used as a tool to reveal functionally relevant residues. By preferentially modifying certain side chains in the POI, chemical probes can uncover new binding motifs to investigate. Here we describe methods for protein modification, and how some pitfalls in the field can be turned into tools to reveal and exploit druggable pockets. Thus, allowing the design of innovative inhibitors against disease-relevant POIs. We discuss methodologies for site-specific modification of lysine, tryptophan, cysteine, histidine and tyrosine and comment on instances where the modified residues were used as targets for functionalization or drug design.

A transthyretin-related protein is functionally expressed in Herbaspirillum seropedicae.

Transthyretin-related proteins (TRPs) constitute a family of proteins structurally related to transthyretin (TTR) and are found in a large range of bacterial, fungal, plant, invertebrate, and vertebrate species. However, it was recently recognized that both prokaryotic and eukaryotic members of this family are not functionally related to transthyretins. TRPs are in fact involved in the purine catabolic pathway and function as hydroxyisourate hydrolases. An open reading frame encoding a protein similar to the Escherichia coli TRP was identified in Herbaspirillum seropedicae genome (Hs_TRP). It was cloned, overexpressed in E. coli, and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein, and circular dichroism spectrum indicated a predominance of beta-sheet structure, as expected for a TRP. We have demonstrated that Hs_TRP is a 5-hydroxyisourate hydrolase and by site-directed mutagenesis the importance of three conserved catalytic residues for Hs_TRP activity was further confirmed. The production of large quantities of this recombinant protein opens up the possibility of obtaining its 3D-structure and will help further investigations into purine catabolism.

Enhancement of the Anti-Aggregation Activity of a Molecular Chaperone Using a Rationally Designed Post-Translational Modification.

Protein behavior is closely regulated by a plethora of post-translational modifications (PTMs). It is therefore desirable to develop approaches to design rational PTMs to modulate specific protein functions. Here, we report one such method, and we illustrate its successful implementation by potentiating the anti-aggregation activity of a molecular chaperone. Molecular chaperones are a multifaceted class of proteins essential to protein homeostasis, and one of their major functions is to combat protein misfolding and aggregation, a phenomenon linked to a number of human disorders. In this work, we conjugated a small-molecule inhibitor of the aggregation of α-synuclein, a process associated with Parkinson's disease (PD), to a specific cysteine residue on human Hsp70, a molecular chaperone with five free cysteines. We show that this regioselective conjugation augments in vitro the anti-aggregation activity of Hsp70 in a synergistic manner. This Hsp70 variant also displays in vivo an enhanced suppression of α-synuclein aggregation and its associated toxicity in a Caenorhabditis elegans model of PD.

S-Nitrosylation of cIAP1 Switches Cancer Cell Fate from TNFα/TNFR1-Mediated Cell Survival to Cell Death.

TNFα is a prominent proinflammatory cytokine and a critical mediator for the development of many types of cancer such as breast, colon, prostate, cervical, skin, liver, and chronic lymphocytic leukemia. Binding of TNFα to TNFR1 can lead to divergent signaling pathways promoting predominantly NF-κB activation but also cell death. We report here that the nitric oxide (NO) donor glyceryl trinitrate (GTN) converts TNFα, generated from immune cells or cancer cells stimulated by chemotherapy, into a prodeath mediator in colon and mammary cancer cells. GTN-mediated S-nitrosylation of cIAP1 on cysteines 571 and 574 inhibited its E3 ubiquitin ligase activity, which in turn reduced Lys63-linked ubiquitination of RIP1 and initiated assembly of a death complex. These findings provide insights into how NO can harness advantageous aspects of inflammation in cancer and provide new therapeutic strategies.Significance: Combination of an NO donor with chemotherapeutic drug-induced TNFα represents a potentially valuable anticancer strategy. Cancer Res; 78(8); 1948-57. ©2018 AACR.

A Water-Bridged Cysteine-Cysteine Redox Regulation Mechanism in Bacterial Protein Tyrosine Phosphatases.

The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus, which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators.

Oxidation and Tyrosine Nitration Induce Structural Changes and Inhibits Plasmodium falciparum Falcipain-2 Activity In Vitro.

Falcipain-2 (FP2) is an important hemoglobinase from the malaria parasite Plasmodium falciparum and a suitable target for the development of an antimalarial chemotherapy. Many reports have indicated that radical nitrogen species (RNS) including nitric oxide (NO) are inhibitors of P. falciparum growth and promoters of recovery from malaria symptoms. In this scenario, FP2 emerges as a potential target of RNS, since its inhibition partially hinders the parasite growth. We report that in vitro FP2 did not undergo S-nitrosylation when exposed to the NO-donor GSNO. However, it was modified by a combined mechanism of methionine oxidation and tyrosine nitration in response to SIN-1, and NaNO2- H2O2 treatment. The treatments with the nitrating agents caused a pronounced decrease in protease activity most likely induced by a disruption on the secondary and tertiary structure of FP2. Our data also demonstrate that at least four tyrosine residues were nitrated and found on the surface of the enzyme, partially or completely exposed to the solvent. Although performed in vitro, these results suggest that falcipain-2 may be a target of RNS activity and its inhibition could explain the hindering of the parasite growth when exposed to these radicals. The understanding of the molecular mechanisms involving free radicals and its inhibition activity towards FP2 may be effective in the development of antimalarial therapies.

Site-selective installation of BASHY fluorescent dyes to Annexin V for targeted detection of apoptotic cells.

Fluorophores are indispensable for imaging biological processes. We report the design and synthesis of azide-tagged boronic acid salicylidenehydrazone (BASHY) dyes and their use for site-selective labelling of Annexin V. The Annexin V-BASHY conjugate maintained function and fluorescence as demonstrated by the targeted detection of apoptotic cells.

Protein S-nitrosylation: specificity and identification strategies in plants.

The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the biotin switch technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed.

Proteomic analysis of four Brazilian MON810 maize varieties and their four non-genetically-modified isogenic varieties.

Profiling techniques have been suggested as a nontargeted approach to detect unintended effects in genetically modified (GM) plants. Seedlings from eight Brazilian maize varieties, four MON810 GM varieties and four non-GM isogenic varieties, were grown under controlled environmental conditions. Physiological parameters (aerial part weight, main leaf length, and chlorophyll and total protein contents) were compared, and some differences were observed. Nevertheless, these differences were not constant among all GM and non-GM counterparts. Leaf proteomic profiles were analyzed using two-dimensional gel electrophoresis (2DE) coupled to mass spectrometry, using six 2DE gels per variety. The comparison between MON810 and its counterpart was limited to qualitative differences of fully reproducible protein spot patterns. Twelve exclusive proteins were observed in two of four maize variety pairs; all of these leaf proteins were variety specific. In this study, MON810 leaf proteomes of four varieties were similar to non-GM counterpart leaf proteomes.

Biochemical and structural characterization of two site-directed mutants of Staphylococcus xylosus lipase.

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42 degrees C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80 degrees C all enzymes retained their initial activities.

Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase.

Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.

Cloning, expression, purification, and characterization of a novel esterase from Lactobacillus plantarum.

Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His(6)-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40 degrees C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.