Progettazione ed Analisi Conformazionale di Peptidi ad Attività Antimicrobica.

La presenza di agenti patogeni costituisce un problema di vecchia data nell’insorgenza di infezioni di varia tipologia; pertanto nel corso del tempo è accresciuto l’interesse verso forme batteriche, funginee, virali, al fine di circoscrivere o debellare le infezioni da essi causate. I farmaci antibatterici rappresentano la classe di farmaci più utilizzata, il cui impiego è stato però caratterizzato da un uso eccessivo ed errato, tale da provocare l’insorgenza di patogeni resistenti agli antibiotici e da richiedere lo sviluppo di nuovi farmaci. In tale ottica il presente lavoro di ricerca ha riguardato lo studio del comportamento conformazionale, mediante tecniche spettroscopiche (CD e NMR) e simulazioni di dinamica molecolare, dei seguenti peptidi antimicrobici: Temporina L, Temporina A ed i derivati dell’ormone α-MSH. Sulla base dei risultati conformazionali ottenuti per questi peptidi la seconda parte del progetto si è focalizzata essenzialmente sul design razionale e sull’analisi conformazionale di nuovi analoghi. Sia l’attività biologica che il comportamento conformazionale di tali analoghi sono risultati in accordo con la progettazione effettuata, rappresentando il punto di partenza per lo sviluppo di altre interessanti molecole potenzialmente utilizzabili nelle terapie come agenti anti-infettivi o, eventualmente, in qualità di conservanti alimentari. Tale progettazione ha inoltre portato ad alcuni derivati della Temporina L con elevata attività antimicrobica (antibatterica e/o antifungina) e con ridotta tossicità (valutata come effetto emolitico)

Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins

Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3. DOI: http://dx.doi.org/10.7554/eLife.16673.00

Structure-function relationships and conformational properties of alpha-MSH(6-13) analogues with candidacidal activity

alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogenous linear tridecapeptide with potent anti-inflammatory effects. We firstly demonstrated that alpha-MSH and its C-terminal sequence Lys-Pro-Val [alpha-MSH(11-13)] have antimicrobial effects against two major and representative pathogens: Staphylococcus aureus and Candida albicans. Successively, in an attempt to improve the candidacidal activity of alpha-MSH and to better understand the peptide structure-antifungal activity relations, we have recently designed and synthesized novel peptide analogues. We focused on the sequence alpha-MSH(6-13), which contains the invariant melanocortin core sequence His-Phe-Arg-Trp (6-9) and also contains the sequence Lys-Pro-Val (11-13) important for antimicrobial activity. In that structure-activity study, we discovered several compounds that have greater candidacidal activity than alpha-MSH, among which the peptide [D-Nal-7,Phe-12]-alpha-MSH(6-13) was the most potent. Here, we report a detailed conformational analysis by spectroscopic and computational methods of three peptides, alpha-MSH(6-13) (1), [D-Nal-7,Phe-12]-alpha-MSH(6-13) (2) and [D-Nal-7,Asp-12]-alpha-MSH(6-13) (3). Peptides were chosen on the basis of their candidacidal activities and were studied in membrane mimetic environment (SDS micelles). Different turn structures were observed for the three peptides and a conformation-activity model was developed based on these results. This study offers a structural basis for the design of novel peptide and non-peptide analogues to be used as new antimicrobial agent

Alanine scanning analysis and structure–function relationships of the frog-skin antimicrobial peptide temporin-1Ta

The increasing resistance of bacteria and fungi to the available antibiotic/antimycotic drugs urges for a search for new anti-infective compounds with new modes of action. In line of this, natural CAMPs represent promising and attractive candidates. Special attention has been devoted to frog-skin temporins, because of their short size (10-14 residues long) and their unique features. In particular, temporin-1Ta has the following properties: (i) it is mainly active on Gram-positive bacteria; (ii) it can synergize, when combined with temporin-1Tl, in inhibiting both gram-negative bacterial growth and the toxic effect of LPS; (iii) it preserves biological activity in the presence of serum; and (iv) it is practically not hemolytic. Rational design of CAMPs represents a straightforward approach to obtain a peptide with a better therapeutic index. Here, we used alanine scanning analogs to elucidate the contribution of the side chains of each amino acid residue to the peptide's antimicrobial and hemolytic activity. Beside providing insight into the biophysical attributes and the critical positions within the peptide sequence, which govern the antimicrobial/hemolytic activity of this temporin isoform, our studies assist in optimizing the design of temporin-based lead structures for the production of new anti-infective agents

Structure-activity relationship, conformational and biological studies of temporin L analogues

Temporins are naturally occurring peptides with promising features, which could lead to the development of new drugs. Temporin-1Tl (TL) is the strongest antimicrobial peptide, but it is toxic on human erythrocytes and this fact makes the design of synthetic analogues with a higher therapeutic index vital.We studied the structure-activity relationships of a library of TL derivatives focusing on the correlation between the α-helix content of the peptides, the nature of their cationic residues, and their antibacterial/antiyeast/hemolytic activities. We found that the percentage of helicity of TL analogues is directly correlated to their hemolytic activity but not to their antimicrobial activity. In addition, we found that the nature of positively charged residues can affect the biological properties of TL without changing the peptide's helicity. It is noteworthy that a single amino acid substitution can prevent the antimicrobial activity of TL, making it a lytic peptide presumably due to its self-association. Last, we identified a novel analogue with properties that make it an attractive topic for future research

author response mitochondrial bol1 and bol3 function as assembly factors for specific iron sulfur proteins

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Author response: Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins

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