New Histopathological Findings in Scarring Alopecias.

CONTEXT.­: Clinical, dermoscopic, and histological diagnostic criteria may overlap in cases with scarring and nonscarring alopecia, making diagnosis difficult for clinicians and pathologists. New histopathological discoveries indicate that the cutaneous adnexal structural and homeostatic unit made up of the pilosebaceous unit, eccrine sweat gland coils (ESGCs), and dermal white adipose tissue may have a role in hair follicle renewal. OBJECTIVE.­: To verify the presence of adipose tissue in the dermis at the level of the isthmus, infiltrating the bundles of the arrector pili muscle in biopsies from the scalp of 3 scarring alopecias: frontal fibrosing alopecia (FFA), fibrosing alopecia in a pattern distribution (FAPD), and lichen planopilaris (LPP). DESIGN.­: We performed a retrospective and descriptive survey of 71 female scalp biopsies from 2016 to 2022 diagnosed at the Dermatopathology Laboratory at Fluminense Federal University of Rio de Janeiro. Two pathologists reviewed and diagnosed the cases correlating pathological features with clinical and dermoscopic findings. RESULTS.­: The histopathological findings of adipose tissue infiltration in the dermis at the level of the isthmus and in the bundles of the arrector pili muscle, and the displacement of ESGCs were more frequently identified in FFA, followed by FAPD and less frequently found in LPP. CONCLUSIONS.­: According to our research, adipose tissue infiltration in the dermis at the level of the isthmus and in the bundles of the arrector pili muscle, and the displacement of ESGCs were observed in 3 scarring alopecias (FFA, FAPD, and LPP) and seems to be involved in the development of scarring alopecia.

Desferrioxamine biosynthesis: diverse hydroxamate assembly by substrate-tolerant acyl transferase DesC.

Hydroxamate groups play key roles in the biological function of diverse natural products. Important examples include trichostatin A, which inhibits histone deacetylases via coordination of the active site zinc(II) ion with a hydroxamate group, and the desferrioxamines, which use three hydroxamate groups to chelate ferric iron. Desferrioxamine biosynthesis in Streptomyces species involves the DesD-catalysed condensation of various N-acylated derivatives of N-hydroxycadaverine with two molecules of N-succinyl-N-hydroxycadaverine to form a range of linear and macrocyclic tris-hydroxamates. However, the mechanism for assembly of the various N-acyl-N-hydroxycadaverine substrates of DesD from N-hydroxycadaverine has until now been unclear. Here we show that the desC gene of Streptomyces coelicolor encodes the acyl transferase responsible for this process. DesC catalyses the N-acylation of N-hydroxycadaverine with acetyl, succinyl and myristoyl-CoA, accounting for the diverse array of desferrioxamines produced by S. coelicolor The X-ray crystal structure of DesE, the ferrioxamine lipoprotein receptor, in complex with ferrioxamine B (which is derived from two units of N-succinyl-N-hydroxycadaverine and one of N-acetyl-N-hydroxycadaverine) was also determined. This showed that the acetyl group of ferrioxamine B is solvent exposed, suggesting that the corresponding acyl group in longer chain congeners can protrude from the binding pocket, providing insights into their likely function. This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms.

The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops.

Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70.

HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner.

Structural basis for acyl acceptor specificity in the achromobactin biosynthetic enzyme AcsD.

Siderophores are known virulence factors, and their biosynthesis is a target for new antibacterial agents. A non-ribosomal peptide synthetase-independent siderophore biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii achromobactin biosynthesis protein D (AcsD) enzyme has been shown to enantioselectively esterify citric acid with l-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme-bound citryl adenylate by l-serine to produce the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of l-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the non-ribosomal peptide synthetase-independent siderophore superfamily may have biotransformation potential.

The Scottish Structural Proteomics Facility: targets, methods and outputs.

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.

The long-overlooked enzymology of a nonribosomal peptide synthetase-independent pathway for virulence-conferring siderophore biosynthesis.

Siderophores are high-affinity ferric iron chelators biosynthesised and excreted by most microorganisms that play an important role in iron acquisition. Siderophore-mediated scavenging of ferric iron from hosts contributes significantly to the virulence of pathogenic microbes. As a consequence siderophore biosynthesis is an attractive target for chemotherapeutic intervention. Two main pathways for siderophore biosynthesis exist in microbes. One pathway involves nonribosomal peptide synthetase (NRPS) multienzymes while the other is NRPS-independent. The enzymology of NRPS-mediated siderophore biosynthesis has been extensively studied for more than a decade. In contrast, the enzymology of NRPS-independent siderophore (NIS) biosynthesis was overlooked for almost thirty years since the first genetic characterisation of the NIS biosynthetic pathway to aerobactin. However, the past three years have witnessed an explosion of interest in the enzymology of NIS synthetases, the key enzymes in the assembly of siderophores via the NIS pathway. The biochemical characterisation of ten purified recombinant synthetases has been reported since 2007, along with the first structural characterisation of a synthetase by X-ray crystallography in 2009. In this feature article we summarise the recent progress that has been made in understanding the long-overlooked enzymology of NRPS-independent siderophore biosynthesis, highlight important remaining questions, and suggest likely directions for future research.

Chapter 17. Siderophore biosynthesis a substrate specificity assay for nonribosomal peptide synthetase-independent siderophore synthetases involving trapping of acyl-adenylate intermediates with hydroxylamine.

Siderophores are an important group of structurally diverse natural products that play key roles in ferric iron acquisition in most microorganisms. Two major pathways exist for siderophore biosynthesis. One is dependent on nonribosomal peptide synthetase (NRPS) multienzymes. The enzymology of several NRPS-dependent pathways to structurally diverse siderophores has been intensively studied for more than 10 years and is generally well understood. The other major pathway is NRPS-independent. It relies on a novel family of synthetase enzymes that until recently has received very little attention. Over the last 2 years, these enzymes have begun to be intensively investigated and several examples have now been characterized. In this article, we give an overview of the enzymology of NRPS-dependent and NRPS-independent pathways for siderophore biosynthesis, using selected examples to highlight key features. An important facet of many studies of the enzymology of siderophore biosynthesis has been to investigate the substrate specificity of the synthetase enzymes involved. For NRPS-dependent pathways, the ATP-pyrophophate exchange assay has been widely used to investigate the substrate specificity of adenylation domains within the synthetase multienzymes. This assay is ineffective for NRPS-independent siderophore (NIS) synthetases, probably because pyrophosphate is not released from the enzyme after the carboxylic acid substrate and ATP react to form an acyl adenylate. An alternative assay for enzymes that form acyl adenylates involves trapping of the activated carboxyl group with hydroxylamine to form a hydroxamic acid that can be converted to its ferric complex and detected spectrophotometrically. This assay has not been widely used for NRPS adenylation domains. Here, we show that it is an effective assay for examining the carboxylic acid substrate specificity of NIS synthetases. Application of the assay to the type B NIS synthetase AcsA shows that it is selective for alpha-ketoglutaric acid, confirming a bioinformatics-based prediction of the substrate specificity of this enzyme.

AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis.

Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent siderophore (NIS) pathway, which relies on a different family of sparsely investigated synthetases. Here we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, an NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryladenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a unique acyladenylate-forming enzyme with a new fold and chemical catalysis strategy.

Purification, crystallization and data collection of Pectobacterium chrysanthemi AcsD, a type A siderophore synthetase.

AcsD, a type A siderophore synthetase with a molecular weight of 71 140 Da from Pectobacterium chrysanthemi, has been expressed, purified and crystallized at 293 K. The protein crystallized in the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 80.3, b = 95.7, c = 161.1 A, alpha = beta = gamma = 90 degrees . Systematic absences were consistent with space group P2(1)2(1)2(1). A complete data set has been collected to 2.25 A resolution on BM14 at the ESRF. Consideration of the likely solvent content suggested that the asymmetric unit contained two molecules. Gel-filtration experiments indicated that the protein was a dimer, although self-rotation analyses did not detect a convincing twofold symmetry axis in the asymmetric unit. The protein has no convincing sequence match to any known structure and thus solution is likely to require experimental phasing.

Bisucaberin biosynthesis: an adenylating domain of the BibC multi-enzyme catalyzes cyclodimerization of N-hydroxy-N-succinylcadaverine.

The bisucaberin biosynthetic gene cluster has been identified in Vibrio salmonicida and a domain from within the BibC multienzyme encoded by the cluster has been shown to catalyse ATP-dependent dimerisation and macrocyclisation of N-hydroxy-N-succinylcadaverine to form bisucaberin.

Petrobactin biosynthesis: AsbB catalyzes condensation of spermidine with N8-citryl-spermidine and its N1-(3,4-dihydroxybenzoyl) derivative.

The AsbB enzyme, which is involved in the biosynthesis of the virulence-conferring siderophore petrobactin in Bacillus anthracis, is shown to catalyze efficient ATP-dependent condensation of spermidine, but not N1-(3,4-dihydroxbenzoyl)-spermidine, with N8-citryl-spermidine or N1-(3,4-dihydroxbenzoyl)-N8-citryl-spermidine, suggesting that N1-(3,4-dihydroxbenzoyl)-spermidine is very unlikely to be a significant intermediate in petrobactin biosynthesis, contrary to previous suggestions.

Identification of a gene cluster that directs putrebactin biosynthesis in Shewanella species: PubC catalyzes cyclodimerization of N-hydroxy-N-succinylputrescine.

Putrebactin is a dihydroxamate iron chelator produced by the metabolically versatile marine bacterium Shewanella putrefaciens. It is a macrocyclic dimer of N-hydroxy-N-succinyl-putrescine (HSP) and is structurally related to desferrioxamine E, which is a macrocyclic trimer of N-hydroxy-N-succinyl-cadaverine (HSC). We recently showed that DesD, a member of the NIS synthetase superfamily, catalyzes the key step in desferrioxamine E biosynthesis: ATP-dependent trimerisation and macrocylization of HSC. Here we report identification of a conserved gene cluster in the sequenced genomes of several Shewanella species, including Shewanella putrefaciens, which is hypothesized to direct putrebactin biosynthesis from putrescine, succinyl-CoA and molecular oxygen. The pubC gene within this gene cluster encodes a protein with 65% similarity to DesD. We overexpressed pubC from Shewanella species MR-4 and MR-7 in E. coli. The resulting His6-PubC fusion proteins were purified by Ni-NTA affinity and gel filtration chromatography. The recombinant proteins were shown to catalyze ATP-dependent cyclodimerization of HSP to form putrebactin. The uncyclized dimer of HSP pre-putrebactin was shown to be an intermediate in the conversion of two molecules of HSP to putrebactin. The data indicate that pre-putrebactin is converted to putrebactin via PubC-catalyzed activation of the carboxyl group by adenylation, followed by PubC-catalyzed nucleophilic attack of the amino group on the carbonyl carbon of the acyl adenylate. This mechanism for macrocycle formation is very different from the mechanism involved in the biosynthesis of many other macrocyclic natural products, where already-activated acyl thioesters are converted by thioesterase domains of polyketide synthases and nonribosomal peptide synthetases to macrocycles via covalent enzyme bound intermediates. The results of this study demonstrate that two closely related enzymes, PubC and DesD, catalyze specific cyclodimerization and cyclotrimerization reactions, respectively, of structurally similar substrates, raising intriguing questions regarding the molecular mechanism of specificity.

A new family of ATP-dependent oligomerization-macrocyclization biocatalysts.

Oligomerization and macrocyclization reactions are key steps in the biosynthesis of many bioactive natural products. Important macrocycles include the antibiotic daptomycin (1; ref. 1), the immunosuppressant FK-506 (2; ref. 2), the anthelmintic avermectin B1a (3; ref. 3) and the insecticide spinosyn A (4; ref. 4); important oligomeric macrocycles include the siderophores enterobactin (5; ref. 5) and desferrioxamine E (6; ref. 6). Biosynthetic oligomerization and macrocyclization reactions typically involve covalently tethered intermediates and are catalyzed by thioesterase domains of polyketide synthase and nonribosomal peptide synthetase multienzymes. Here we report that the purified recombinant desferrioxamine siderophore synthetase DesD from Streptomyces coelicolor M145 catalyzes ATP-dependent trimerization-macrocyclization of a chemically synthesized 10-aminocarboxylic acid substrate via noncovalently bound intermediates. DesD is dissimilar to other known synthetase families but is similar to other enzymes known or proposed to be required for the biosynthesis of omega-aminocarboxylic acid-derived cyclodimeric siderophores. This suggests that DesD is the first biochemically characterized member of a new family of oligomerizing and macrocyclizing synthetases.

Avaliação da Podofilotoxina 0, 15 por cento na terapia de lesões de HPV / Evaluation of podofilitoxina 0, 15 per cent for treatment of HPV lesions

Introdução: o condiloma acuminado é provocado pelo Papilomavírus humano (HPV), do qual existem mais de 100 tipos distintos. Acredita-se que seja um dos facilitadores da transmissão sexual do HIV, sendo por isso importante o seu diagnóstico precoce e tratamento. Comumente utilizados em saúde pública, o ácido tricloroacético (TCA 90 por cento) e a eletrocauterização são métodos dolorosos e que necessitam de formação médica especializada. A solução de podofilina 25 por cento é bastante utilizada, porém a preparação é extremamente instável com curto período de durabilidade. Objetivo: os autores estudaram o uso domiciliar da podofilotoxina creme a 0,15 por cento, principal substância ativa da resina de podofilina, que inibe a metáfase celular, sendo uma medicação antiviral, que pode ser auto-aplicada. É considerada como forma de tratamento praticamente indolor quando comparada aos demais métodos. Métodos: os pacientes foram orientados a aplicarem o medicamento duas vezes ao dia, três vezes por semana por um máximo de quatro semanas (ciclos), com avaliação do número de lesões por consulta após cada ciclo. Resultados: a análise estatística provou ser significativa a diminuição do número de lesões ao longo do tratamento. O número de lesões da primeira consulta comparado respectivamente com o número após o primeiro ciclo e após o quarto ciclo mostraram p-valor=0,001134 e p-valor=0,000699. Ao final do quarto ciclo, observou-se cura em 72 por cento, melhora em 15 por cento e inalterado em 13 por cento dos pacientes. Conclusão: a auto-aplicação de podofilotoxina creme a 0,15 por cento para o tratamento de condiloma acuminado mostrou-se como um dos métodos mais eficazes para tratamento do HPV.

Enzymatic synthesis of aroma compound xylosides using transfer reaction by Trichoderma longibrachiatum xylanase.

Enzymatic synthesis of aroma compound xylosides was performed by Trichoderma longibrachiatum xylanase. Information concerning the nature of xylosides present in the reaction medium was obtained by GC-EI-MS, by GC-NCI-MS of TFA derivatives, and by positive FAB-MS of the reaction mixtures. Moreover, the structures of isolated benzyl beta-D-xylopyranoside and 4-O-beta-xylopyranosyl-beta-D-xylopyranoside were established by (1)H and (13)C NMR and heteronuclear two-dimensional ((1)H-(13)C) chemical shift correlation. The results obtained for hexyl and benzyl alcohol xylosides indicated that a reaction implying a transfer of one to two or three xylose units from xylan was involved. The enzyme was able to recognize xylobiose, xylotriose, and xylan as xylose donors. Benzyl xyloside, produced independently of xylobioside and xylotrioside, was found as the major kinetic product of the reaction. Benzyl xyloside was produced in higher quantities and at a higher rate than that obtained for the di- and trixyloside derivatives. The maximum production for benzyl xyloside, 1.29 g/L, was obtained in the presence of hexane (50%) used as cosolvent. Xylosides and xylobiosides of several aroma compounds, (Z)-hex-3-en-1-ol, heptan-2-ol, geraniol, nerol, and citronellol, were synthesized in different amounts, from 850 mg/L for (Z)-hex-3-en-1-yl xylosides to 1.5 mg/L for citronellyl xylosides. No synthesis occurred when menthol, linalool, and eugenol were used as acceptors.