O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications

The Synthesis of Kynurenic Acid in Mammals: An Updated Kynurenine Aminotransferase Structural KATalogue

Kynurenic acid (KYNA) is a bioactive compound that is produced along the kynurenine pathway (KP) during tryptophan degradation. In a few decades, KYNA shifted from being regarded a poorly characterized by-product of the KP to being considered a main player in many aspects of mammalian physiology, including the control of glutamatergic and cholinergic synaptic transmission, and the coordination of immunomodulation. The renewed attention being paid to the study of KYNA homeostasis is justified by the discovery of selective and potent inhibitors of kynurenine aminotransferase II, which is considered the main enzyme responsible for KYNA synthesis in the mammalian brain. Since abnormally high KYNA levels in the central nervous system have been associated with schizophrenia and cognitive impairment, these inhibitors promise the development of novel anti-psychotic and pro-cognitive drugs. Here, we summarize the currently available structural information on human and rodent kynurenine aminotransferases (KATs) as the result of global efforts aimed at describing the full complement of mammalian isozymes. These studies highlight peculiar features of KATs that can be exploited for the development of isozyme-specific inhibitors. Together with the optimization of biochemical assays to measure individual KAT activities in complex samples, this wealth of knowledge will continue to foster the identification and rational design of brain penetrant small molecules to attenuate KYNA synthesis, i.e., molecules capable of lowering KYNA levels without exposing the brain to the harmful withdrawal of KYNA-dependent neuroprotective actions

First thermostable CLIP-tag by rational design applied to an archaeal O6-alkyl-guanine-DNA-alkyl-transferase

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O6-benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC8. The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC8 was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O6-alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag varian

The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate

SNAP-tag ® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag ® and its relative thermostable version (SsOGT-H5 ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag

Nutrition and IBD: Malnutrition and/or Sarcopenia? A Practical Guide

Malnutrition is a major complication of inflammatory bowel disease (IBD). This mini review is focusing on main determinants of malnutrition in IBD, the most important components of malnutrition, including lean mass loss and sarcopenia, as an emerging problem. Each one of these components needs to be well considered in a correct nutritional evaluation of an IBD patient in order to build a correct multidisciplinary approach. The review is then focusing on possible instrumental and clinical armamentarium for the nutritional evaluation

Longevidade das restaurações posterior com resina composta e amálgama

Nos últimos anos temos assistido a um grande desenvolvimento dos materiais restauradores, bem como a uma maior exigência estética quer dos dentistas, quer dos próprios pacientes relativamente às restaurações dentárias. O requisito estético por parte dos pacientes levou à necessidade do desenvolvimento de materiais restauradores utilizados em Medicina Dentária e à inevitavel substituição do amálgama de prata por resinas compostas. Foi feita uma revisão da literatura avaliar as caraterísticas e as limitações do amálgama de prata e das resinas compostas, bem como na comparação da sua utilização em restaurações de dentes posteriores. O amálgama foi de facto o material mais utilizado no decurso do século passado, mas caiu em desuso por Médicos Dentistas devido à sua aparência e ao desenvolvimento de novos materiais restauradores à cor de dente, com sucesso clínico claro e verificável em restaurações posteriores. Apesar do seu baixo custo, da sua boa aderência e da sua durabilidade servirem como vantagens significativas, o amálgama tem algumas desvantagens, tais como a baixa estética e a necessidade de fazer grandes preparações dentárias, o que prejudica a estrutura do dente saudável. Outro problema é a presença de mercúrio que, apesar da falta de estudos conclusivos sobre a sua toxicidade, é um tópico que gera muita controvérsia entre dentistas e pacientes. Para superar esses problemas, têm sido desenvolvidas as resinas compostas. No entanto, estes materiais também têm tido algumas limitações, tais como a contração de polimerização, e a sensibilidade por microinfiltrações marginais, com subsequente desenvolvimento das cáries secundárias. Portanto, é importante para o dentista reexaminar as necessidades específicas de cada paciente e ter em conta as indicações e contra-indicações de cada material, de acordo com a situação clínica e os objetivos do paciente, a fim de decidir qual o material a utilizar para fazer restaurações em dentes posteriore

Mycobacterium tuberculosis Pathogenesis, Infection Prevention and Treatment

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis (MTB) and it represents a persistent public health threat for a number of complex biological and sociological reasons. According to the most recent Global Tuberculosis Report (2019) edited by the World Health Organization (WHO), TB is considered the ninth cause of death worldwide and the leading cause of mortality by a single infectious agent, with the highest rate of infections and death toll rate mostly concentrated in developing and low-income countries. We present here the editorial section to the Special Issue entitled \u201cMycobacterium tuberculosis Pathogenesis, Infection Prevention and Treatment\u201d that includes 7 research articles and a review. The scientific contributions included in the Special Issue mainly focus on the characterization of MTB strains emerging in TB endemic countries as well as on multiple mechanisms adopted by the bacteria to resist and to adapt to antitubercular therapies

Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair

Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), an ancient disease which still today causes 1.4 million deaths worldwide per year. Long-term, multi-agent anti-tubercular regimens can lead to the anticipated non-compliance of the patient and increased drug toxicity, which in turn can contribute to the emergence of drug-resistant MTB strains that are not susceptible to first- and second-line available drugs. Hence, there is an urgent need for innovative antitubercular drugs and vaccines. A number of biochemical processes are required to maintain the correct homeostasis of DNA metabolism in all organisms. Here we focused on reviewing our current knowledge and understanding of biochemical and structural aspects of relevance for drug discovery, for some such processes in MTB, and particularly DNA synthesis, synthesis of its nucleotide precursors, and processes that guarantee DNA integrity and genome stability. Overall, the area of drug discovery in DNA metabolism appears very much alive, rich of investigations and promising with respect to new antitubercular drug candidates. However, the complexity of molecular events that occur in DNA metabolic processes requires an accurate characterization of mechanistic details in order to avoid major flaws, and therefore the failure, of drug discovery approaches targeting genome integrity

Structure of Thermococcus litoralis \u3941-pyrroline-2-carboxylate reductase in complex with NADH and L-proline

L-Hydroxyproline (L-Hyp) is a nonstandard amino acid that is present in certain proteins, in some antibiotics and in the cell-wall components of plants. L-Hyp is the product of the post-translational modification of protein prolines by prolyl hydroxylase enzymes, and the isomers trans-3-hydroxy-L-proline (T3LHyp) and trans-4-hydroxy-L-proline (T4LHyp) are major components of mammalian collagen. T4LHyp follows two distinct degradation pathways in bacteria and mammals, while T3LHyp is metabolized by a two-step metabolic pathway that is conserved in bacteria and mammals, which involves a T3LHyp dehydratase and a \u3941-pyrroline-2-carboxylate (Pyr2C) reductase. In order to shed light on the structure and catalysis of the enzyme involved in the second step of the T3LHyp degradation pathway, the crystal structure of Pyr2C reductase from the archaeon Thermococcus litoralis DSM 5473 complexed with NADH and L-proline is presented. The model allows the mapping of the residues involved in cofactor and product binding and represents a valid model for rationalizing the catalysis of Pyr2C reductases