Marek Lisowski

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti-inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well-defined conformation in methanol, its structure was distinct from the corresponding 50-59 fragment in the native ubiquitin molecule. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 423-431, 2009.

Isoform-specific variation in the intrinsic disorder of the ecdysteroid receptor N-terminal domain.

The Drosophila melanogaster ecdysteroid receptor (EcR) is a member of the nuclear hormone receptor superfamily. EcR controls animal development and metamorphosis by activating or repressing the transcription of target genes. There are three EcR isoforms, EcRA, EcRB1, and EcRB2 that exhibit diverse spatial and temporal distributions within various tissues and reveal essential functional differences. These differences can be attributed to the isoform‐specific N‐terminal domains (NTDs), which differ in length and primary structure. To lay a foundation for understanding of the molecular mechanism underlying functional diversity of the isoforms, we have carried out a comprehensive biochemical and biophysical analysis of purified hexahistidine‐tagged EcRA and EcRB1 NTDs (EcRA‐NTD and EcRB1‐NTD). The results, along with in silico examinations of the primary structures indicate that the EcR NTDs exhibit properties of premolten globule‐like intrinsically disordered proteins. Furthermore, we demonstrate for the first time that NTDs of isoforms of a particular nuclear hormone receptor exhibit distinct structural properties. In silico analysis revealed that the EcRA‐NTD sequence has a bigger tendency for disorder than the EcRB1‐NTD sequence. Accordingly, the circular dichroism experiments demonstrated that EcRA‐NTD has lower regular secondary structure content than EcRB1‐NTD and the size‐exclusion chromatography showed that EcRA‐NTD is less compact than EcRB1‐NTD. Furthermore, the limited proteolysis analysis revealed that the C‐terminal region common to both NTDs is more susceptible to the enzymatic cleavage in EcRA‐NTD than in EcRB1‐NTD. We postulate that unique conformational states of EcRA‐NTD and EcRB1‐NTD might act as the starting points for the functional diversity of EcRA and EcRB1 isoforms. Proteins 2009.

Starmaker exhibits properties of an intrinsically disordered protein.

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.

Ovine colostrum nanopeptide affects amyloid beta aggregation

A colostral proline‐rich polypeptide complex (PRP) consisting of over 30 peptides shows beneficial effects in Alzheimers disease (AD) patients when administered in the form of sublinqual tablets called Colostrinin. The aim of the present studies was to investigate whether nanopeptide fragment of PRP (NP) – one of the PRP complex components can affect aggregation of amyloid β (Aβ1‐42). The effect of NP on Aβ aggregation was studied using Thioflavin T (ThT) binding, atomic force microscopy, and analyzing circular dichroism spectra. Results presented suggest that NP can directly interact with amyloid beta, inhibit its aggregation and disrupt existing aggregates acting as a β sheet breaker and reduce toxicity induced by aggregated forms of Aβ.

A dramatic change in the interaction of Cu(II) with bio-peptides promoted by SDS--a model for complex formation on a membrane surface.

The extent of complex formation between Cu(II) and many biologically active oligopeptides has been shown to change significantly in the presence of SDS micelles, a recognized model for cell lipid membranes. Protonation constants of peptides can be increased by up to 2 log unit, especially when they contain hydrophobic side chains. Metal complex formation is generally less extensive and the conformations of peptides can be altered dramatically when compared to those in simple aqueous solution.

Lipid-binding role of βII-spectrin ankyrin-binding domain

It is known that erythroid and non‐erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially‐expressed recombinant peptides representing βII(brain)‐spectrins ankyrin‐binding domain and its truncated mutants showed lipid‐binding activity, although only those containing a full‐length amino terminal fragment showed high to moderate affinity towards phospholipid mono‐ and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on βI‐spectrins ankyrin‐binding domain [Hryniewicz‐Jankowska A, et al. Mapping of ankyrin‐sensitive, PE/PC mono‐ and bilayer binding site in erythroid beta‐spectrin. Biochem J 2004;382:677–85]. Moreover, we tested also the effect of transient transfection of living cells of several cell‐lines with vectors coding for GFP‐conjugates including βII and also βI full‐length ankyrin‐binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full‐length ankyrin‐binding domain of βII and βI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin‐binding domain truncated at their N‐terminal region. Our results therefore indicate the importance of N‐terminal region for lipid‐binding activity of the β‐spectrin ankyrin‐binding domain and its substantial role in maintaining the spectrin‐based skeleton distribution.

N-[tert-Butoxy­carbonyl­glycyl-(Z)-α,β-de­hydro­phenyl­alanyl­glycyl-(E)-α,β-de­hydro­phenyl­alanyl]­glycine methyl ester dihydrate

The title pentapeptide, Boc0—Gly1–ΔZPhe2—Gly3–ΔEPhe4—Gly5—OMe, C30H35N5O8·2H2O, adopts the type I β-turn conformation for the ΔZPhe2—Gly3 residues. It is stabilized by a 4\rightarrow1 intramolecular hydrogen bond between the ΔEPhe4 NH and Gly1 CO groups. All the amino acid residues in the pentapeptide sequence are linked trans to each other. The crystal structure is stabilized by intra- and intermolecular hydrogen bonds.

A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline

We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established as cyclo(PPFFILLX), where X is a non-proteinaceous amino acid, N-methyl-4-aminoproline. This amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanar cis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysed cis/trans amide-Pro bond isomerisation.

Stereochemistry of thiazolidine ring formation from aminals and cysteine

Abstract Aminals derived from Z-S-Ala, Z-R-Ala and Pht-S-Ala were coupled with R- and S-cysteine to give thiazolidine analogues of dipeptides with the configuration of the newly formed stereogenic carbon depending on the alanine configuration. 1 H-NMR and CD spectra were measured to check the configurational homogeneity of the products, and NOESY spectra were used to assign a configuration of the new stereogenic centre.

Conformational Investigations in the Tuftsin Group

In our recent review,‘ the problem of the biologically active conformation of tuftsin was discussed. Existing models of this conformationz4 all suggest a folding of the molecule. The type of folding is different, however, with every proposition. The recent results of the investigations of the activity of some tuftsin analogues make each of the proposed models questionable. A distinct activity of cyclo-tuftsin, synthesized by Chippens,’ contradicts our 8-bend model. On the other hand, the inhibitory potency of Gluz-tuftsin reported by Najjar,6 and also the very high phagocytosis-stimulating activity found by Bar-Shavit et al.’ for NHz-terminal substance P tetrapeptide Arg-Pro-Lys-Pro, argue against Nikiforovich-Chippens’s model because in both these tetrapeptides, the structure suggested by Nikiforovich is not likely. Thus, the question of the conformation of biologically active tuftsin remains open. We do not know if the tuftsin receptor recognizes the conformation of the main chain of peptide, or the precise amino acid sequence. There exist no data concerning the contact areas responsible for peptide-receptor interaction. There is, however, much evidence on the solution conformation of tuftsin analogues. Our attempt is to provide a review of this evidence, and especially the data obtained in our laboratory. Our own investigations of this subject were connected mainly with two problems: