Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein†1 (HYOU1) Suppressing Pathogenic Fibroblast Activation.

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation. The study encompasses medicinal chemistry, molecular phenotyping assays, chemoproteomics, bulk RNA-sequencing analysis, target validation experiments, and chemical absorption, distribution, metabolism, excretion and toxicity (ADMET)/pharmacokinetic (PK)/in vivo evaluation. The parallel synthesis employed for the production of the new benzamide derivatives enabled us to a) pinpoint key structural elements of the scaffold that provide potent fibroblast-deactivating effects in cells, b) discriminate atoms or groups that favor or disfavor a desirable ADMET profile, and c) identify metabolic "hot spots". Furthermore, we report the discovery of the first-in-class inhibitor leads for hypoxia up-regulated protein 1 (HYOU1), a member of the heat shock protein 70 (HSP70) family often associated with cellular stress responses, particularly under hypoxic conditions. Targeting HYOU1 may therefore represent a potentially novel strategy to modulate fibroblast activation and treat chronic inflammatory and fibrotic disorders.

Substrate Specificity of the Highly Thermostable Esterase EstDZ3.

Esterases are among the most studied enzymes, and their applications expand into several branches of industrial biotechnology. Yet, despite the fact that information on their substrate specificity is crucial for selecting or designing the best fitted biocatalyst for the desired application, it cannot be predicted from their amino acid sequence. In this work, we studied the substrate scope of the newly discovered hydrolytic extremozyme, EstDZ3, against a library of esters with variable carbon chain lengths in an effort to understand the crucial amino acids for the substrate selectivity of this enzyme. EstDZ3 appears to be active against a wide range of esters with high selectivity towards medium- to long-carbon chain vinyl esters. In-silico studies of its 3D structure revealed that the selectivity might arise from the mainly hydrophobic nature of the active site's environment.

SuptoxD2.0: A second-generation engineered Escherichia coli strain achieving further enhanced levels of recombinant membrane protein production.

The bacterium Escherichia coli is among the most popular hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently generated the specialized MP-producing E. coli strain SuptoxD, which upon co-expression of the effector gene djlA, is capable of alleviating two major bottlenecks in bacterial recombinant MP production: it suppresses the toxicity that frequently accompanies the MP-overexpression process and it markedly increases the cellular accumulation of membrane incorporated and properly folded recombinant MP. Combined, these two positive effects result in dramatically enhanced volumetric yields for various recombinant MPs of both prokaryotic and eukaryotic origin. Based on the observation that djlA is found in the genomes of various pathogenic bacteria, the aim of the present work was to investigate (a) whether other naturally occurring DjlA variants can exert the MP toxicity-suppressing and production-promoting effects similarly to the E. coli DjlA and (b) if we can identify a DjlA variant whose efficiency surpasses that of the E. coli DjlA of SuptoxD. We report that a quite surprisingly broad variety of homologous DjlA proteins exert beneficial effects on recombinant MP when overexpressed in E. coli. Furthermore, we demonstrate that the Salmonella enterica DjlA is an even more potent enhancer of MP productivity compared with the E. coli DjlA of SuptoxD. Based on this, we constructed a second-generation SuptoxD strain, termed SuptoxD2.0, whose MP-production capabilities surpass significantly those of the original SuptoxD, and we anticipate that SuptoxD2.0 will become a broadly utilized expression host for recombinant MP production in bacteria.

Activity and specificity studies of the new thermostable esterase EstDZ2.

In this paper, we study the activity and specificity of EstDZ2, a new thermostable carboxyl esterase of unknown function, which was isolated from a metagenome library from a Russian hot spring. The biocatalytic reaction employing EstDZ2 proved to be an efficient method for the hydrolysis of aryl p-, o- or m-substituted esters of butyric acid and esters of secondary alcohols. Docking studies revealed structural features of the enzyme that led to activity differences among the different substrates.

Escherichia coli strains with precise domain deletions in the ribonuclease RNase E can achieve greatly enhanced levels of membrane protein production.

Escherichia coli is one of the most widely utilized hosts for production of recombinant membrane proteins (MPs). Bacterial MP production, however, is usually accompanied by severe toxicity and low-level volumetric accumulation. In previous work, we had discovered that co-expression of RraA, an inhibitor of the RNA-degrading activity of RNase E, can efficiently suppress the cytotoxicity associated with the MP overexpression process and, simultaneously, enhance significantly the cellular accumulation of membrane-incorporated recombinant MPs in bacteria. Based on this, we constructed the specialized MP-producing E. coli strain SuptoxR, which can achieve dramatically enhanced volumetric yields of well-folded recombinant MPs. Ιn the present work, we have investigated whether domain deletions in the E. coli RNase E, which exhibit reduced ribonucleolytic activity, can result in suppressed MP-induced toxicity and enhanced recombinant MP production, in a manner resembling the conditions of rraA overexpression in E. coli SuptoxR. We have found that some strains encoding specific RNase E truncation variants can achieve significantly enhanced levels of recombinant MP production. Among these, we have found a single RNase E variant strain, which can efficiently suppress MP-induced toxicity and achieve greatly enhanced levels of recombinant MP production for proteins of both prokaryotic and eukaryotic origin. Based on its properties, and in analogy to the original SuptoxR strain, we have termed this strain SuptoxRNE22. E. coli SuptoxRNE22 can perform better than commercially available bacterial strains, which are frequently utilized for recombinant MP production. We anticipate that SuptoxRNE22 will become a widely utilized host for recombinant MP production in bacteria.

Protein aggregation and therapeutic strategies in SOD1- and TDP-43- linked ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with severe socio-economic impact. A hallmark of ALS pathology is the presence of aberrant cytoplasmic inclusions composed of misfolded and aggregated proteins, including both wild-type and mutant forms. This review highlights the critical role of misfolded protein species in ALS pathogenesis, particularly focusing on Cu/Zn superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP-43), and emphasizes the urgent need for innovative therapeutic strategies targeting these misfolded proteins directly. Despite significant advancements in understanding ALS mechanisms, the disease remains incurable, with current treatments offering limited clinical benefits. Through a comprehensive analysis, the review focuses on the direct modulation of the misfolded proteins and presents recent discoveries in small molecules and peptides that inhibit SOD1 and TDP-43 aggregation, underscoring their potential as effective treatments to modify disease progression and improve clinical outcomes.

Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli.

Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR-GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor. Three multi-copy hits were isolated by both methods: nagD, encoding the ribonucleotide phosphatase NagD; a fragment of nlpD, encoding a truncation of the predicted lipoprotein NlpD, and the three-gene cluster ptsN-yhbJ-npr, encoding three proteins of the nitrogen phosphotransferase system. Expression of these genes resulted in a 3- to 10-fold increase in the yields of different mammalian GPCRs. Our data is consistent with the hypothesis that the expression of these genes may serve to maintain the integrity of the bacterial periplasm and to provide a favorable environment for proper membrane protein folding, possibly by inducing a fine-tuned stress response and/or via modifying the composition of the bacterial cell envelope.

The Discovery of Peptide Macrocycle Rescuers of Pathogenic Protein Misfolding and Aggregation by Integrating SICLOPPS Technology and Ultrahigh-Throughput Screening in Bacteria.

The phenomenon of protein misfolding and aggregation has been widely associated with numerous human diseases, such as Alzheimer's disease, systemic amyloidosis and type 2 diabetes, the vast majority of which remain incurable. To advance early stage drug discovery against these diseases, investigation of molecular libraries with expanded diversities and ultrahigh-throughput screening methodologies that allow deeper investigation of chemical space are urgently required. Toward this, we describe how Escherichia coli can be engineered so as to enable (1) the production of expanded combinatorial libraries of short, drug-like, head-to-tail cyclic peptides and (2) their simultaneous functional screening for identifying effective inhibitors of protein misfolding and aggregation using a genetic assay that links protein folding and misfolding to cell fluorescence. In this manner, cyclic peptides with the ability to inhibit pathogenic protein misfolding and/or aggregation can be readily selected by flow cytometric cell sorting in an ultrahigh-throughput fashion. This biotechnological approach accelerates significantly the identification of hit/lead molecules with potentially therapeutic properties against devastating diseases.

Second-Generation Escherichia coli SuptoxR Strains for High-Level Recombinant Membrane Protein Production.

Escherichia coli is one of the most widely utilized hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently engineered a specialized E. coli strain for enhanced recombinant MP production, termed SuptoxR. By appropriately co-expressing the effector gene rraA, SuptoxR can suppress the high toxicity, which is frequently observed during the MP-overexpression process, and, at the same time, enhance significantly the cellular accumulation of membrane-incorporated and properly folded recombinant MP. The combination of these two beneficial effects results in dramatically enhanced volumetric yields for various prokaryotic and eukaryotic MPs. Here, we engineered second-generation SuptoxR strains with further improved properties, so that they can achieve even higher levels of recombinant MP production. We searched for naturally occurring RraA variants with similar or improved MP toxicity-suppressing and production-promoting effects to that of the native E. coli RraA of the original SuptoxR strain. We found that the RraA proteins from Proteus mirabilis and Providencia stuartii can be even more potent enhancers of MP productivity than the E. coli RraA. By exploiting these two newly identified RraAs, we constructed two second-generation SuptoxR strains, termed SuptoxR2.1 and SuptoxR2.2, whose MP-production capabilities often surpass those of the original SuptoxR significantly. SuptoxR2.1 and SuptoxR2.2 are expected to become widely useful expression hosts for recombinant MP production in bacteria.

Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies.

The expression of IgG antibodies in Escherichia coli is of increasing interest for analytical and therapeutic applications. In this work, we describe a comprehensive and systematic approach to the development of a dicistronic expression system for enhanced IgG expression in E. coli encompassing: (i) random mutagenesis and high-throughput screening for the isolation of over-expressing strains using flow cytometry and (ii) optimization of translation initiation via the screening of libraries of synonymous codons in the 5' region of the second cistron (heavy chain). The effects of different promoters and co-expression of molecular chaperones on full-length IgG production were also investigated. The optimized system resulted in reliable expression of fully assembled IgG at yields between 1 and 4 mg/L of shake flask culture for different antibodies.

Strain engineering for improved expression of recombinant proteins in bacteria.

Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins.

Simple genetic selection protocol for isolation of overexpressed genes that enhance accumulation of membrane-integrated human G protein-coupled receptors in Escherichia coli.

The efficient production of membrane proteins in bacteria remains a major challenge. In this work, we sought to identify overexpressed genes that enhance the yields of recombinant membrane proteins in Escherichia coli. We developed a genetic selection system for bacterial membrane protein production, consisting of membrane protein fusions with the enzyme beta-lactamase and facile selection of high-production strains on ampicillin-containing media. This system was used to screen the ASKA library, an ordered library of plasmids encoding all the known E. coli open reading frames (ORFs), and several clones with the ability to accumulate enhanced amounts of recombinant membrane proteins were selected. Notably, coexpression of ybaB, a gene encoding a putative DNA-binding protein of unknown function, was found to enhance the accumulation of a variety of membrane-integrated human G protein-coupled receptors and other integral membrane proteins in E. coli by up to 10-fold. The results of this study highlight the power of genetic approaches for identifying factors that impact membrane protein biogenesis and for generating engineered microbial hosts for membrane protein production.

High-level Production of Recombinant Membrane Proteins Using the Engineered Escherichia coli Strains SuptoxD and SuptoxR.

We have previously described the development of two specialized Escherichia coli strains for high-level recombinant membrane protein (MP) production. These engineered strains, termed SuptoxD and SuptoxR, are capable of suppressing the cytotoxicity caused by MP overexpression and of producing greatly enhanced MP yields. Here, we present a Bio-protocol that describes gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains.

XynDZ5: A New Thermostable GH10 Xylanase.

Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4-ß-xylanase that performs optimally at 65-75°C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (ß/α)8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.

Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor.

The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production.

Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli.

BACKGROUND: The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity. RESULTS AND CONCLUSION: In this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.

Optimization of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR.

Membrane proteins (MPs) execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium Escherichia coli has served as a very popular overexpression host for biochemical/structural studies of membrane proteins as well. Bacterial recombinant membrane protein production, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low levels of final biomass and minute volumetric yields. In previous work, we generated the engineered E. coli strains SuptoxD and SuptoxR, which upon coexpression of the effector genes djlA or rraA, respectively, can suppress the cytotoxicity caused by MP overexpression and produce enhanced MP yields. Here, we systematically looked for gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains. We have found that, under optimal conditions, SuptoxD and SuptoxR achieve greatly enhanced recombinant production for a variety of MP, irrespective of their archaeal, eubacterial, or eukaryotic origin. Furthermore, we demonstrate that the use of these engineered strains enables the production of well-folded recombinant MPs of high quality and at high yields, which are suitable for functional and structural studies. We anticipate that SuptoxD and SuptoxR will become broadly utilized expression hosts for recombinant MP production in bacteria.

ANASTASIA: An Automated Metagenomic Analysis Pipeline for Novel Enzyme Discovery Exploiting Next Generation Sequencing Data.

Metagenomic analysis of environmental samples provides deep insight into the enzymatic mixture of the corresponding niches, capable of revealing peptide sequences with novel functional properties exploiting the high performance of next-generation sequencing (NGS) technologies. At the same time due to their ever increasing complexity, there is a compelling need for ever larger computational configurations to ensure proper bioinformatic analysis, and fine annotation. With the aiming to address the challenges of such an endeavor, we have developed a novel web-based application named ANASTASIA (automated nucleotide aminoacid sequences translational plAtform for systemic interpretation and analysis). ANASTASIA provides a rich environment of bioinformatic tools, either publicly available or novel, proprietary algorithms, integrated within numerous automated algorithmic workflows, and which enables versatile data processing tasks for (meta)genomic sequence datasets. ANASTASIA was initially developed in the framework of the European FP7 project HotZyme, whose aim was to perform exhaustive analysis of metagenomes derived from thermal springs around the globe and to discover new enzymes of industrial interest. ANASTASIA has evolved to become a stable and extensible environment for diversified, metagenomic, functional analyses for a range of applications overarching industrial biotechnology to biomedicine, within the frames of the ELIXIR-GR project. As a showcase, we report the successful in silico mining of a novel thermostable esterase termed "EstDZ4" from a metagenomic sample collected from a hot spring located in Krisuvik, Iceland.

Bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation.

Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer's and Parkinson's diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid-ß peptide (Aß42), linked to Alzheimer's disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce Aß42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

Engineered systems for detection and discovery of nuclear hormone-like compounds.

Biomolecular engineering has many applications in the identification of potentially therapeutic compounds. An important class of these compounds is those that bind and modulate the activity of the human nuclear hormone receptors (NHRs). NHRs are typically made up of clearly defined domains with known function, including one that mediates ligand recognition and NHR activation. Engineered systems that include these ligand-binding domains (LBDs) can be used to identify potential therapeutic ligands that target a given NHR. These methods must couple the binding event to a readily detectable signal, ideally in a high-throughput format. Recent efforts have delivered a variety of new techniques, including those that involve fusions of LBDs to easily assayed reporter proteins. In some cases these systems allow hormone-dependent selectable phenotypes to be generated in non-native hosts, providing potential tools for both isolation and evolution of new therapeutics in vivo. Here we provide an overview and a comparison of many of the available tools in this area, with an emphasis on a novel allosteric hormone-regulated sensor protein that provides ligand-dependent phenotypes in the relatively simple background of Escherichia coli bacterial cells.