Elżbieta Jankowska

Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C.

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.

Copper(II) binding by fragments of α-synuclein containing M1-D2- and -H50-residues; a combined potentiometric and spectroscopic study

Stability constants and ligand donor sets of the copper(II) complexes of the NH2-29-56(L1)(AA30GKTKEGVLYV40GSKTKEGVVH50GVATVA56-NH2), NH2-M29-D30-56(L2) and Ac-M29-D30-56(L3) fragments of alpha-synuclein were determined in aqueous solution for 1 : 1 metal-to-ligand molar ratio in the pH range 2.5-10.5. The tyrosine residue in the 39th position of the alpha-synuclein fragments does not take part in the coordination of the metal ion. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the L3 fragment compared to that of the L2 peptide. When the amino group is blocked (L3) the imidazole nitrogen of the histidine residue acts as an anchoring site and at higher pH the 3N {N(Im),2N-} and 4N {N(Im),3N-} complexes are formed. The L1 peptide at physiological pH forms in equilibrium 3N {NH2,N-,CO,N(Im)} and 4N {NH2,2N-,N(Im)} complexes. For the L2 peptide the coordination of the copper(II) ions starts from the N-terminal Met residue and with increasing of pH the Asp residue in second position of amino acid sequence coordinates and stabilizes significantly the 2N complex as a result of chelation through the beta-carboxylate group. At physiological pH the 3N {NH2,N-,beta-COO-,N(Im)} coordination mode dominates. At pH above 6 the results for the L2 fragment suggest the formation of 3N and 4N complexes (in equatorial plane) and the involvement of the lateral NH2 group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra sigma (epsilon-NH2-Lys) --> Cu(II) charge transfer transition is observed. The stability constants for the L2 fragment of alpha-synuclein of the 4N {NH2,2N-,N(Im)} and {NH2,3N-} complexes are higher about 1.5 and 0.7 orders of magnitude, respectively, by comparison to those of the L1 peptide. This increase may be explained by the involvement of the epsilon-NH2 group of Lys residue in the coordination sphere of metal ion.

The Proteasome in Health and Disease

The giant proteolytic factory called the proteasome came a long way from a biochemical curio to a major regulator of cellular physiology and a renowned drug target within the ubiquitin proteasome pathway (UPP). Thanks to availability of highly specific inhibitors of the proteasome, in less than twenty years it was possible to identify major transcription factors, cyclins, and products of oncogenes as crucial substrates for the UPP. Nine years passed since the FDA speedily approved bortezomib, the inhibitor of proteasome, for treatment of multiple myeloma. One year after its approval, the field was honored by awarding the Nobel Prize to Hershko, Ciechanover and Rose for introducing the concept of controlled proteolysis of ubiquitin-tagged substrates, with proteasome as the intracellular recycling facility. Taking into consideration the universal involvement of the proteasome in the life of all cells in human body, it comes to no surprise that the enzyme is deeply implicated in etiology, progression, diagnosis or cure of multiple diseases. Below we discuss some aspects of the involvement: from direct causative links to changes in proteasome properties that correlate with pathological conditions. We start with diseases collectively known as cancer, and with immune system-related pathologies. Here, the proteasome inhibitors are either already used in clinics, or undergo advanced preclinical screening. Then, we will continue with cardiovascular disorders, followed by aging. Changes of the proteasome make-up during aging may be a priming factor for neurodegenerative diseases, described last. We discuss the potential for proteasome regulation: inhibition, activation or specificity modulation, to successfully enter the clinical setting.

Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C

Human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of papain‐like mammalian proteases, has been produced in its full‐length form by recombinant techniques and crystallized in two polymorphic forms: cubic and tetragonal. A selenomethionyl derivative of the protein, obtained by Escherichia coli expression and with complete Met→Se‐Met substitution confirmed by mass spectrometry, amino‐acid analysis and X‐ray absorption spectra, was crystallized in the cubic form. A truncated variant of the protein, lacking ten N‐terminal residues, has also been crystallized. The crystals of this variant are tetragonal and, like the two polymorphs of the full‐length protein, contain multiple copies of the molecule in the asymmetric unit, suggesting oligomerization of the protein.

Coordination abilities of neurokinin A and its derivative and products of metal-catalyzed oxidation

The classical tachykinins, substance P, neurokinin A and neurokinin B are predominantly found in the nervous system where they act as neurotransmitters and neuromodulators. Significantly reduced levels of these peptides were observed in neurodegenerative diseases and it may be suggested that this reduction may also result from the copper(II)-catalyzed oxidation. The studies of the interaction of copper(II) with neurokinin A and the copper(II)-catalyzed oxidation were performed. Copper(II) complexes of the neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH(2)) and acetyl-neurokinin A (Ac-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH(2)) were studied by potentiometric, UV-Vis (UV-visible), CD (circular dichroism) and EPR spectroscopic methods to determine the stoichiometry, stability constants and coordination modes in the complexes formed. The histidine residue in first position of the peptide chain of neurokinin A coordinates strongly to Cu(II) ion with histamine-like {NH(2), N(Im)} coordination mode. With increasing of pH, the formation of a dimeric complex Cu(2)H(2)L(2) was found but this dimeric species does not prevent the deprotonation and coordination of the amide nitrogens. In the Ac-neurokinin A case copper(II) coordination starts from the imidazole nitrogen of the His; afterwards three deprotonated amide nitrogens are progressively involved in copper coordination. To elucidate the products of the copper(II)-catalyzed oxidation of the neurokinin A and Ac-neurokinin A, liquid chromatography-mass spectrometry (LC-MS) method and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. Oxidation target for both studied peptides is the histidine residue coordinated to the metal ions. Both peptides contain Met and His residues and are very susceptible on the copper(II)-catalyzed oxidation.

Potential allosteric modulators of the proteasome activity

Proteasome, consisting of a tube-shaped proteolytic core particle and attached to it regulatory modules, is a multifunctional enzymatic complex essential for the ubiquitin-proteasome metabolic pathway. Due to its immense involvement in regulation of cellular physiology, the proteasome is an acknowledged anticancer drug target and potential target to treat inflammatory or degenerative diseases. So far, competitive inhibitors of the core particle gain most consideration as drugs. We postulate that noncompetitively-acting small-molecule compounds would provide excellent means to precisely regulate actions of the proteasome. In this study, we evaluated five short peptides based on sequences of two proteins known to interact with the core proteasome: HIV-1 Tat and PA28/REG activator. We performed Circular Dichroism (CD), Fourier Transformed Infrared Spectroscopy (FTIR), and Nuclear Magnetic Resonance (NMR) analysis, supplemented by MD simulations, and tested influence of the peptides on performance of the core particle active sites and functioning of regulatory modules. We found that PP2-containing Tat peptides are noncompetitive inhibitors of the core, interfering with the actions of PA28alphabeta activator. In addition, at low concentrations the turn-prone Tat2 is able to activate the latent core. The random coil-structured PA28-derived peptides display only weak or nondetectable direct effects on the core activities, exhibiting, however, a positive cooperation with activity-enhancing actions of PA28alphabeta.

Zn(II) ions bind very efficiently to tandem repeat region of "prion related protein" (PrP-rel-2) of zebra-fish. MS and potentiometric evidence.

Multi-histidine peptide fragments of zebra-fish prion protein are effective ligands for Zn(II) ions. Moreover the formation of a dinuclear complex species with a longer peptide can suggest the existence of the cooperative effect in the metal ion binding.

Influence of point mutations on the stability, dimerization, and oligomerization of human cystatin C and its L68Q variant

Human cystatin C (hCC) is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently losing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that the mechanism of this process is based on the three dimensional swapping of the protein domains. In our work we have focused on the impact of two proposed “hot spots” in cystatin C structure on its conformational stability. Encouraged by promising results of the theoretical calculations, we designed and produced several hCC hinge region point mutation variants that display a variety of conformational stability and propensity for dimerization and aggregation. A similar approach, i.e., rational mutagenesis, has been also applied to study the amyloidogenic L68Q variant to determine the contribution of hydrophobic interactions and steric effect on the stability of monomeric cystatin C. In this overview we would like to summarize the results of our studies. The impact of a particular mutation on the properties of the studied proteins will be presented in the context of their thermal and mechanical stability, in vitro dimerization tendency as well as the outcome of crystallization. Better understanding of the mechanism and, especially, factors affecting conformational stability of cystatin C and access to stable monomeric and dimeric versions of the protein opens new perspectives in explaining the role of dimers and the domain swapping process in hCC oligomerization, as well as designing potential inhibitors of this process.

Complexation abilities of neuropeptide gamma toward copper(II) ions and products of metal-catalyzed oxidation.

The stability constants, stoichiometry, and solution structures of copper(II) complexes of neuropeptide gamma (NPG) (D(1)-A-G-H(4)-G-Q-I-S-H(9)-K-R-H(12)-K-T-D-S-F-V-G-L-M(21)-NH(2)) and acethyl-neuropeptide gamma (Ac-D(1)-A-G-H(4)-G-Q-I-S-H(9)-K-R-H(12)-K-T-D-S-F-V-G-L-M(21)-NH(2)) were determined in aqueous solution. For both peptides the additional deprotonations were observed; therefore, the potentiometric data calculations for NPG were only made in 2.5-7.4 pH range. For Ac-NPG one additional deprotonation was observed, likely hydroxy group of Ser residue, and the potentiometric data calculations in the 2.5-10.5 pH range may be performed. The potentiometric and spectroscopic data (UV-vis, CD, EPR) for the neuropeptide gamma show that a D(1) residue stabilizes significantly the copper(II) complexes with 1N {NH(2),β-COO(-)}, 2N {NH(2),β-COO(-),N(Im)}, and 3N {NH(2),β-COO(-),2N(Im)} coordination modes as the result of coordination through the β-carboxylate group. The Ac-NPG forms with the copper(II) ions the 3N {3N(Im)} complex in a wide 4.5-7.5 pH range. At higher pH deprotonation and coordination of the sequential amide nitrogens occur. Metal-catalyzed oxidation of proteins is mainly a site-specific process in which amino acids at metal-binding sites to the protein are preferentially oxidized. To elucidate the products of the copper(II)-catalyzed oxidation of NPG and Ac-NPG the liquid chromatography-mass spectrometry method (LC-MS) and the Cu(II)/H(2)O(2) as a model oxidizing system were employed. For solutions containing a 1:4 peptide-hydrogen peroxide molar ratio oxidation of the methionine residue to methionine sulphone was observed. For the 1:1:4 Cu(II)-NPG-H(2)O(2) system oxidation of two His residues and cleavage of the G(3)-H(4) and R(11)-H(12) peptide bonds were detected, supporting involvement of His(4) and His(12) in binding of the copper(II) ions. Oxidations of three histidine residues to 2-oxohistidines and fragmentations of Ac-NPG near the His (H(4), H(9),H(12)) residues support participation of the histidyl-imidazole nitrogen atoms in coordination of the metal ions.

Specific cathepsin B inhibitor is cell-permeable and activates presentation of TTC in primary human dendritic cells

Cathepsins of the cysteine, aspartyl, and serine classes are involved in antigen processing in the class II major histocompatibility complex (MHC) loading compartment. Investigation of these proteases in living cells is difficult to perform due to the lack of highly specific cell-permeable inhibitors. Recently, a highly selective cathepsin B (CatB) inhibitor, Z-Arg-Leu-Arg-alpha-aza-glycyl-Ile-Val-OMe (ZRLR), was described. We found that ZRLR is cell-permeable and specifically inhibits CatB, in contrast to the CatB inhibitor, CA074-OMe, which blocks cysteine cathepsins in addition to CatB in primary human antigen-presenting cells (APC). Furthermore, we compared both CA074-OMe and ZRLR in the ability to alter tetanus toxin C-fragment (TTC) presentation to T cells by different APC. As a result, we found enhanced presentation of TTC in the presence of ZRLR, as determined by detection of pro-inflammatory cytokines. We conclude that ZRLR is a specific, cell-permeable CatB inhibitor which can be used for antigen presenting studies in situ.