Targeted Strategies for Degradation of Key Transmembrane Proteins in Cancer.

Targeted protein degradation is an attractive technology for cancer treatment due to its ability to overcome the unpredictability of the small molecule inhibitors that cause resistance mutations. In recent years, various targeted protein degradation strategies have been developed based on the ubiquitin-proteasome system in the cytoplasm or the autophagy-lysosomal system during endocytosis. In this review, we describe and compare technologies for the targeted inhibition and targeted degradation of the epidermal growth factor receptor (EGFR), one of the major proteins responsible for the onset and progression of many types of cancer. In addition, we develop an alternative strategy, called alloAUTO, based on the binding of new heterocyclic compounds to an allosteric site located in close proximity to the EGFR catalytic site. These compounds cause the targeted degradation of the transmembrane receptor, simultaneously activating both systems of protein degradation in cells. Damage to the EGFR signaling pathways promotes the inactivation of Bim sensor protein phosphorylation, which leads to the disintegration of the cytoskeleton, followed by the detachment of cancer cells from the extracellular matrix, and, ultimately, to cancer cell death. This hallmark of targeted cancer cell death suggests an advantage over other targeted protein degradation strategies, namely, the fewer cancer cells that survive mean fewer chemotherapy-resistant mutants appear.

Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery.

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, ß, ß' and ω are simultaneously coexpressed. Appending a 3' UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase).

D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

Targeting Degradation of EGFR through the Allosteric Site Leads to Cancer Cell Detachment-Promoted Death.

Targeting epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors (TKI) has been widely exploited to disrupt aberrant phosphorylation flux in cancer. However, a bottleneck of potent TKIs is the acquisition of drug resistance mutations, secondary effects, and low ability to attenuate tumor progression. We have developed an alternative means of targeting EGFR that relies on protein degradation through two consecutive routes, ultimately leading to cancer cell detachment-related death. We describe furfuryl derivatives of 4-allyl-5-[2-(4-alkoxyphenyl)-quinolin-4-yl]-4H-1,2,4-triazole-3-thiol that bind to and weakly inhibit EGFR tyrosine phosphorylation and induce strong endocytic degradation of the receptor in cancer cells. The compound-promoted depletion of EGFR resulted in the sequestration of non-phosphorylated Bim, which no longer ensured the integrity of the cytoskeleton machinery, as shown by the detachment of cancer cells from the extracellular matrix (ECM). Of particular note, the longer CH3(CH2)n chains in the terminal moiety of the anti-EGFR molecules confer higher hydrophobicity in the allosteric site located in the immediate vicinity of the catalytic pocket. Small compounds accelerated and enhanced EGFR and associated proteins degradation during EGF and/or glutamine starvation of cultures, thereby demonstrating high potency in killing cancer cells by simultaneously modulating signaling and metabolic pathways. We propose a plausible mechanism of anti-cancer action by small degraders through the allosteric site of EGFR. Our data represent a rational and promising perspective in the treatment of aggressive tumors.

Reactive Chemicals and Electrophilic Stress in Cancer: A Minireview.

Exogenous reactive chemicals can impair cellular homeostasis and are often associated with the development of cancer. Significant progress has been achieved by studying the macromolecular interactions of chemicals that possess various electron-withdrawing groups and the elucidation of the protective responses of cells to chemical interventions. However, the formation of electrophilic species inside the cell and the relationship between oxydative and electrophilic stress remain largely unclear. Derivatives of nitro-benzoxadiazole (also referred as nitro-benzofurazan) are potent producers of hydrogen peroxide and have been used as a model to study the generation of reactive species in cancer cells. This survey highlights the pivotal role of Cu/Zn superoxide dismutase 1 (SOD1) in the production of reactive oxygen and electrophilic species in cells exposed to cell-permeable chemicals. Lipophilic electrophiles rapidly bind to SOD1 and induce stable and functionally active dimers, which produce excess hydrogen peroxide leading to aberrant cell signalling. Moreover, reactive oxygen species and reactive electrophilic species, simultaneously generated by redox reactions, behave as independent entities that attack a variety of proteins. It is postulated that the binding of the electrophilic moiety to multiple proteins leading to impairing different cellular functions may explain unpredictable side effects in patients undergoing chemotherapy with reactive oxygen species (ROS)-inducing drugs. The identification of proteins susceptible to electrophiles at early steps of oxidative and electrophilic stress is a promising way to offer rational strategies for dealing with stress-related malignant tumors.

Rapid Diminution in the Level and Activity of DNA-Dependent Protein Kinase in Cancer Cells by a Reactive Nitro-Benzoxadiazole Compound.

The expression and activity of DNA-dependent protein kinase (DNA-PK) is related to DNA repair status in the response of cells to exogenous and endogenous factors. Recent studies indicate that Epidermal Growth Factor Receptor (EGFR) is involved in modulating DNA-PK. It has been shown that a compound 4-nitro-7-[(1-oxidopyridin-2-yl)sulfanyl]-2,1,3-benzoxadiazole (NSC), bearing a nitro-benzoxadiazole (NBD) scaffold, enhances tyrosine phosphorylation of EGFR and triggers downstream signaling pathways. Here, we studied the behavior of DNA-PK and other DNA repair proteins in prostate cancer cells exposed to compound NSC. We showed that both the expression and activity of DNA-PKcs (catalytic subunit of DNA-PK) rapidly decreased upon exposure of cells to the compound. The decline in DNA-PKcs was associated with enhanced protein ubiquitination, indicating the activation of cellular proteasome. However, pretreatment of cells with thioglycerol abolished the action of compound NSC and restored the level of DNA-PKcs. Moreover, the decreased level of DNA-PKcs was associated with the production of intracellular hydrogen peroxide by stable dimeric forms of Cu/Zn SOD1 induced by NSC. Our findings indicate that reactive oxygen species and electrophilic intermediates, generated and accumulated during the redox transformation of NBD compounds, are primarily responsible for the rapid modulation of DNA-PKcs functions in cancer cells.

Activation of EGFR by small compounds through coupling the generation of hydrogen peroxide to stable dimerization of Cu/Zn SOD1.

Activation of cell signaling by reactive chemicals and pollutants is an important issue for human health. It has been shown that lipophilic nitro-benzoxadiazole (NBD) compounds rapidly move across the plasma membrane and enhance Epidermal Growth Factor Receptor (EGFR) tyrosine phosphorylation in cancer cells. Unlike ligand-dependent activation, the mechanism of this induction relies on the generation of hydrogen peroxide, which is involved in the activation of the catalytic site of the receptor and the inactivation of protein tyrosine phosphatase PTP-1B. Production of H2O2 during redox transformation of NBD compounds is associated with the transition of a monomeric form of Cu/Zn superoxide dismutase 1 (SOD1) to stable dimers. The highly stable and functionally active SOD1 dimer, in the absence of adequate activities in downstream reactions, promotes the disproportionate production and accumulation of intracellular hydrogen peroxide shortly after exposure to NBD compounds. The intrinsic fluorescence of small compounds was used to demonstrate their binding to SOD1. Our data indicate that H2O2 and concomitantly generated electrophilic intermediates behave as independent entities, but all contribute to the biological reactivity of NBD compounds. This study opens a promising path to identify new biomarkers of oxidative/electrophilic stress in the progression of cancer and other diseases.

TP53 transcription factor for the NEDD9/HEF1/Cas-L gene: potential targets in Non-Small Cell Lung Cancer treatment.

Lung cancer is a serious public health problem. Although there has been significant progress in chemotherapy, non-small cell lung cancer is still resistant to current treatments, primarily because of the slow rate of cell development. It is thus important to find new molecules directed against targets other than proliferation agents. Considering the high proportion of mutant proteins in tumor cells, and the high rate of mutation of the TP53 gene in all cancers, and in NSCLC in particular, this gene is a perfect target. Certain new molecules have been shown to restore the activity of mutated p53 protein, for example PRIMA-1, which reactivates the His273 mutant p53. In a previous study, we presented triazine A190, a molecule with a cytostatic activity that blocks cells in the G1 phase and induces apoptosis. Here, we show that A190 not only restores mutant p53 activity, but also induces an overexpression of the NEDD9 gene, leading to apoptotic death. These findings might offer hope for the development of new targeted therapies, specific to tumor cells, which spare healthy cells.

Screening and discovery of nitro-benzoxadiazole compounds activating epidermal growth factor receptor (EGFR) in cancer cells.

Peptide ligand-induced dimerization of the extracellular region of the epidermal growth factor receptor (sEGFR) is central to the signal transduction of many cellular processes. A small molecule microarray screen has been developed to search for non-peptide compounds able to bind to sEGFR. We describe the discovery of nitro-benzoxadiazole (NBD) compounds that enhance tyrosine phosphorylation of EGFR and thereby trigger downstream signaling pathways and other receptor tyrosine kinases in cancer cells. The protein phosphorylation profile in cells exposed to NBD compounds is to some extent reminiscent of the profile induced by the cognate ligand. Experimental studies indicate that the small compounds bind to the dimerization domain of sEGFR, and generate stable dimers providing allosteric activation of the receptor. Moreover, receptor phosphorylation is associated with inhibition of PTP-1B phosphatase. Our data offer a promising paradigm for investigating new aspects of signal transduction mediated by EGFR in cancer cells exposed to electrophilic NBD compounds.

Assessment of new cationic porphyrin binding to plasma proteins by planar microarray and spectroscopic methods.

Porphyrins have a unique aromatic structure determining particular photochemical properties that make them promising photosensitizers for anticancer therapy. Previously, we synthesized a set of artificial porphyrins by modifying side-chain functional groups and introducing different metals into the core structure. Here, we have performed a comparative study of the binding properties of 29 cationic porphyrins with plasma proteins by using microarray and spectroscopic approaches. The porphyrins were noncovalently immobilized onto hydrogel-covered glass slides and probed to bio-conjugated human and bovine serum albumins, as well as to human hemoglobin. The signal detection was carried out at the near-infrared fluorescence wavelength (800 nm) that enabled the effect of intrinsic visible wavelength fluorescence emitted by the porphyrins tested to be discarded. Competition assays on porphyrin microarrays indicated that long-chain fatty acids (FAs) (palmitic and stearic acids) decrease porphyrin binding to both serum albumin and hemoglobin. The binding affinity of different types of cationic porphyrins for plasma proteins was quantitatively assessed in the absence and presence of FAs by fluorescent and absorption spectroscopy. Molecular docking analysis confirmed results that new porphyrins and long-chain FAs compete for the common binding site FA1 in human serum albumin and meso-substituted functional groups in porphyrins play major role in the modulation of conformational rearrangements of the protein.

A highly sensitive near-infrared fluorescent detection method to analyze signalling pathways by reverse-phase protein array.

The comprehensive and quantitative analysis of the protein phosphorylation patterns in different cellular context is of considerable and general interest. The ability to quantify phosphorylation of discrete signalling proteins in large collections of biological samples would greatly favour the development of systems biology in the field of cell signalling. Reverse-phase protein array (RPPA) potentially represents a very attractive approach to map signal transduction networks with high throughput. In the present report, we describe an improved detection method for RPPA combining near-infrared with one or two rounds of tyramide-based signal amplification. The LOQ was lowered from 6.84 attomoles with a direct detection protocol to 0.21 attomole with two amplification steps. We validated this method in the context of intracellular signal transduction triggered by follicle-stimulating hormone in HEK293 cells. We consistently detected phosphorylated proteins in the sub-attomole range from less than 1 ng of total cell extracts. Importantly, the method correlated with Western blot analysis of the same samples while displaying excellent intra- and inter-slide reproducibility. We conclude that RPPA combined with amplified near-infrared detection can be used to capture the subtle regulations intrinsic to signalling network dynamics at an unprecedented throughput, from minute amounts of biological samples.

Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis.

Dynamin 2 (DNM2) is a large GTPase involved in the release of nascent vesicles during endocytosis and intracellular membrane trafficking. Distinct DNM2 mutations, affecting the middle domain (MD) and the Pleckstrin homology domain (PH), have been identified in autosomal dominant centronuclear myopathy (CNM) and in the intermediate and axonal forms of the Charcot-Marie-Tooth peripheral neuropathy (CMT). We report here the first CNM mutation (c.1948G>A, p.E650 K) in the DNM2 GTPase effector domain (GED), leading to a slowly progressive moderate myopathy. COS7 cells transfected with DNM2 constructs harboring a disease-associated mutation in MD, PH, or GED show a reduced uptake of transferrin and low-density lipoprotein (LDL) complex, two markers of clathrin-mediated receptor endocytosis. A decrease in clathrin-mediated endocytosis was also identified in skin fibroblasts from one CNM patient. We studied the impact of DNM2 mutant overexpression on epidermal growth factor (EGF)-induced extracellular signal-regulated kinase 1 (ERK1) and ERK2 activation, known to be an endocytosis- and DNM2-dependent process. Activation of ERK1/2 was impaired for all the transfected mutants in COS7 cells, but not in CNM fibroblasts. Our results indicate that impairment of clathrin-mediated endocytosis may play a role in the pathophysiological mechanisms leading to DNM2-related diseases, but the tissue-specific impact of DNM2 mutations in both diseases remains unclear.

PTEN contributes to profound PI3K/Akt signaling pathway deregulation in dystrophin-deficient dog muscle.

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3beta (GSK3beta), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38delta and p38gamma kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3beta, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3beta and PTEN activities. PTEN/GSK3beta-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3beta and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.

Triad pattern algorithm for predicting strong promoter candidates in bacterial genomes.

BACKGROUND: Bacterial promoters, which increase the efficiency of gene expression, differ from other promoters by several characteristics. This difference, not yet widely exploited in bioinformatics, looks promising for the development of relevant computational tools to search for strong promoters in bacterial genomes. RESULTS: We describe a new triad pattern algorithm that predicts strong promoter candidates in annotated bacterial genomes by matching specific patterns for the group I sigma70 factors of Escherichia coli RNA polymerase. It detects promoter-specific motifs by consecutively matching three patterns, consisting of an UP-element, required for interaction with the alpha subunit, and then optimally-separated patterns of -35 and -10 boxes, required for interaction with the sigma70 subunit of RNA polymerase. Analysis of 43 bacterial genomes revealed that the frequency of candidate sequences depends on the A+T content of the DNA under examination. The accuracy of in silico prediction was experimentally validated for the genome of a hyperthermophilic bacterium, Thermotoga maritima, by applying a cell-free expression assay using the predicted strong promoters. In this organism, the strong promoters govern genes for translation, energy metabolism, transport, cell movement, and other as-yet unidentified functions. CONCLUSION: The triad pattern algorithm developed for predicting strong bacterial promoters is well suited for analyzing bacterial genomes with an A+T content of less than 62%. This computational tool opens new prospects for investigating global gene expression, and individual strong promoters in bacteria of medical and/or economic significance.

Similarity and divergence between the RNA polymerase alpha subunits from hyperthermophilic Thermotoga maritima and mesophilic Escherichia coli bacteria.

The alpha subunit (alphaTm) of Thermotoga maritima RNA polymerase has been characterized to investigate its role in transcriptional regulation in one of the few known anaerobic hyperthermophilic bacteria. The highly thermostable alphaTm shares 54% similarity with its Escherichia coli analogue (alphaEc). The T. maritima rpoA gene coding the alpha subunit does not complement the thermosensitive rpoA112 mutation of E. coli. However, alphaTm and alphaEc show similar folding patterns as determined by circular dichroism. Purified alphaTm binds to the T. maritima PargGo promoter region (probably to a UP-element) and Arg282 appears to be crucial for DNA binding. The thermostable protein is also able to interact with transcription regulatory proteins, like ArgR from T. neapolitana or CRP from E. coli. These data indicate that the RNA polymerase alpha subunit might play a crucial role in the modulation of gene expression in hyperthermophiles.

Competition on nitrocellulose-immobilized antibody arrays: from bacterial protein binding assay to protein profiling in breast cancer cells.

Large scale comparative evaluation of protein expression requires miniaturized techniques to provide sensitive and accurate measurements of the abundance of molecules present as individual and/or assembled protein complexes in cells. The principle of competition between target molecules for binding to arrayed antibodies has recently been proposed to assess differential expression of numerous proteins with one-color or two-color fluorescence detection methods. To establish the limiting factors and to validate the use of alternative detection for protein profiling, we performed competitive binding assays under different conditions. A model experimental protocol was developed whereby the competitive displacement of multi-subunit bacterial RNA polymerase and/or its subunits was evaluated through binding to subunit-specific immobilized monoclonal antibodies. We show that the difference in physico-chemical properties of unlabeled and labeled molecules significantly affects the performance of one-color detection, whereas epitope inaccessibility in the protein complex can prohibit the assessment of competition by both detection methods. Our data also demonstrate that antibody cross-reactivity, target protein truncation and abundance, as well as the cellular compartment of origin are major factors that affect protein profiling on antibody arrays. The experimental conditions established for prokaryotic proteins were adopted to compare protein profiles in the breast tumor-derived cell lines MDA MB-231 and SKBR3. Competitive displacement was detected and confirmed for a number of proteins using both detection methods; however, we show that overall the two-color method is better suited for accurate expression profile evaluation of a large, complex set of proteins. Antibody array data confirm the functional linkage between the ErbB2 receptor and AP-2 transcription factors in these cell lines and highlight unexpected differences in G1 cyclin expression.

High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

Array assessment of phage-displayed peptide mimics of Human Immunodeficiency Virus type 1 gp41 immunodominant epitope: binding to antibodies of infected individuals.

An array of phage-displayed mimetic peptides representing 18 sequences from the HIV-1 gp41 immunodominant epitope was fabricated on a nitrocellulose membrane. It used to compare the antibody-binding affinity of the peptide and to monitor the immune response of four patients prior to and after the initiation of Highly Active Anti-Retroviral Therapy in parallel assays with several probes using near-infrared fluorescence detection. The proposed multiplexed approach is highly sensitive, consumes less sample and can be used to analyze the immune repertoires of virus-infected individuals.

Recognition molecule associated carbohydrate inhibits postsynaptic GABA(B) receptors: a mechanism for homeostatic regulation of GABA release in perisomatic synapses.

Extracellular matrix molecules are important cues in the shaping of nervous system structure and function. Here, we describe a novel mechanism by which the HNK-1 carbohydrate carried by recognition molecules regulates perisomatic inhibition in the hippocampus. Neutralization of HNK-1 activity by an HNK-1 antibody results in GABA(B) receptor-mediated activation of K(+) currents in CA1 pyramidal cells, which elevates extracellular K(+) concentration and reduces evoked GABA release in perisomatic inhibitory synapses. This mechanism is supported by pharmacological analysis in hippocampal slices and data showing that the HNK-1 carbohydrate binds to GABA(B) receptors and inhibits GABA(B) receptor-activated K(+) currents in a heterologous expression system. We suggest that the HNK-1 carbohydrate is involved in homeostatic regulation of GABA(A) receptor-mediated perisomatic inhibition by suppression of postsynaptic GABA(B) receptor activity.

Hyperthermophilic Thermotoga arginine repressor binding to full-length cognate and heterologous arginine operators and to half-site targets.

The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga