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.16673.001

In this work, the naturally occurring antimicrobial peptides temporin A (TA) and L (TL) are studied by spectroscopic (CD and NMR) techniques and molecular dynamics simulation. We analyzed the interactions of TA and TL with sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelles, which mimic bacterial and mammalian membranes, respectively. In SDS, the peptides prefer a location at the micelle-water interface; in DPC, they prefer a location perpendicular to the micelle surface, with the N-terminus imbedded in the hydrophobic core. TL shows higher propensity, with respect to TA, in forming alpha-helical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles. Hence, we have proposed a different molecular mechanism underlying the antimicrobial and hemolytic activities of the two peptides. We also designed new analogues of TA and TL and found interesting differences in their efficacy against microbial species and human erythrocytes.

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.

Temporins constitute a family of amphipathic alpha-helical antimicrobial peptides (AMPs) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. We have recently investigated two members of this family, temporin A (TA) and temporin L (TL), because of their different spectra of antimicrobial activity and toxicity. Consequently, we developed new analogues with promising biological activities named Pro(3)-TL and Gln(3)-TA. In this work, we performed a detailed NMR analysis of the new analogues in SDS and DPC micelles, which mimic bacterial and mammalian membranes, respectively. NMR studies reveal that strongly hemolytic Gln(3)-TA was in a stable helical conformation along the entire sequence, while weakly hemolytic but antimicrobial Pro(3)-TL showed conformational averaging at the N-terminus. Furthermore, molecular dynamics (MD) simulations on TL and Pro(3)-TL were performed in explicit water and DPC micelles. Simulations indicated that both peptides prefer a location at the micelle-water interface; however, Phe(1) of strongly hemolytic TL was embedded more in depth into DPC, and only TL caused a significant distortion of the micelle shape. By combining NMR and computational analyses, we obtained a molecular-level resolution of the interactions between TL and its analogues with membrane mimicking micelles.

The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.

Circulating autoantibodies have been recognized as disease biomarkers of autoimmune diseases. We have previously disclosed a synthetic glycopeptide that is able to detect specific autoantibodies in sera of patients who are affected by multiple sclerosis (MS). This glycopeptide is characterized by a type I' beta-turn around the minimal epitope Asn(Glc) that allows an efficient exposure of this moiety to antibody interactions in the context of a solid-phase immunoenzymatic assay. With the aim of optimizing the glycopeptide-antibody interactions, we analyze a series of new glycopeptides based on different turn structures. Our results confirm the role of conformation in the recognition and binding of synthetic antigenic probes to MS autoantibodies. Glycopeptide 2, which is characterized by a type I beta-turn around the minimal epitope Asn(Glc), shows the highest antibody affinity (IC50 = 11.8 nM), and thus it appears to be a promising tool for the detection of specific autoantibodies as MS biomarker in patients' sera.

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.

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 agents.