Marcin Mielecki

Novel AlkB Dioxygenases—Alternative Models for In Silico and In Vivo Studies

Background ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1–8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. Methodology and Findings Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB − mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. Conclusions Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.

The participation of phytocystatin TrcC-4 in the activity regulation of EP8, the main prolamin degrading cysteine endopeptidase in triticale seeds

Phytocystatins (PCs) are protein inhibitors of endogenous plant endopeptidases and exogenous pathogen proteinases. We have previously described the protein inhibitor TrcC-4, which is probably involved in the control of protein degradation during triticale seeds germination. The occurrence of the LARFAVXEHN motif supports the TrcC-4 designation as a PC. In this paper TrcC-4 was expressed in Escherichia coli using the pET28 expression vector. TrcC-4(6×His) was purified by affinity chromatography with a single step of purification. Western blot analysis showed the presence of TrcC-4 in both developing and germinating triticale seeds. TrcC-4 protein level was higher both in scutellum of germinating seeds and in developing grains of the triticale cultivar more resistant to pre-harvest sprouting (Zorro) than in a less resistant one (Disco). Furthermore it was demonstrated that the activity of EP8, cysteine endopeptidase responsible for the mass hydrolysis of prolamin during germination, is inhibited by TrcC-4(6×His), as confirmed by native PAGE with gliadin as a substrate. These results suggest that phytocystatin TrcC-4 controls the activity of cysteine endopeptidases involved in germination and, thus, is potentially involved in pre-harvest sprouting.

Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes.

New surface plasmon resonance (SPR) sensing platforms which consists of copper (II) complexes of a pentetic acid thiol ligand (DPTA-Cu(II)) and of a thiol derivative of dipyrromethene (DPM-Cu(II) created on the surface of gold SPR disc were applied to oriented immobilization of His-tagged Janus kinase 2 (GST-His6-JAK2). This method is based on the covalent bond formation between histidine from a His-tag chain of a protein and Cu(II) centres from the complexes. The kinetic and thermodynamic parameters of the oriented immobilization of GST-His6-JAK2 protein to DPTA-Cu(II) and DPM-Cu(II) complexes attached to the Au surface of a SPR disc were discussed.

Development of novel molecular probes of the Rio1 atypical protein kinase.

The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases. RIO kinases contain a canonical eukaryotic protein kinase fold, but also display several unusual structural features that potentially create opportunity for the design of selective inhibitors. In an attempt to identify structural leads to target the RIO kinases, a series of pyridine caffeic acid benzyl amides (CABA) were tested for their ability to inhibit the autophosphorylation activity of Archeaoglobus fulgidus Rio1 (AfRio1). Screening of a small library of CABA molecules resulted in the identification of four compounds that measurably inhibited AfRio1 activity. Additional biochemical characterization of binding and inhibition activity of these compounds demonstrated an ATP competitive inhibition mode, and allowed identification of the functional groups that result in the highest binding affinity. In addition, docking of the compound to the structure of Rio1 and determination of the X-ray crystal structure of a model compound (WP1086) containing the desired functional groups allowed detailed analysis of the interactions between these compounds and the enzyme. Furthermore, the X-ray crystal structure demonstrated that these compounds stabilize an inactive form of the enzyme. Taken together, these results provide an important step in identification of a scaffold for the design of selective molecular probes to study molecular mechanisms of Rio1 kinases in vitro and in vivo. In addition, it provides a rationale for the future design of potent inhibitors with drug-like properties targeting an inactive form of the enzyme. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro

Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions.

Cinnamic Acid Derivatives as Inhibitors of Oncogenic Protein Kinases--Structure, Mechanisms and Biomedical Effects.

Cinnamic acid belongs to phenolic-acid class of polyphenols, one of the most abundant plant secondary metabolites. These substances are widely studied because of plethora of their biological activities. In particular, their inhibition of protein kinases contributes to the pleiotropic effects in the cell. Protein kinases are essential in controlling cell signaling networks. Selective targeting of oncogenic protein kinases increases clinical anticancer efficacy. Cinnamic acid and related compounds have inspired researchers in the design of numerous synthetic and semisynthetic inhibitors of oncogenic protein kinases for the past three decades. Interest in cinnamoyl-scaffold-containing compounds revived in recent years, which was stimulated by modern drug design and discovery methodologies such as in vitro and in silico HTS. This review presents cinnamic acid derivatives and analogs for which direct inhibition of protein kinases was identified. We also summarize significance of the above protein kinase families - validated or promising targets for anticancer therapies. The inhibition mode may vary from ATP-competitive, through bisubstrate-competitive and mixedcompetitive, to non-competitive one. Kinase selectivity is often correlated with subtle chemical modifications, and may also be steered by an additional non-cinnamoyl fragment of the inhibitor. Specific cinnamic acid congeners may synergize their effects in the cell by a wider range of activities, like suppression of additional enzymes, e.g. deubiquitinases, influencing the same signaling pathways (e.g. JAK2/STAT). Cinnamic acid, due to its biological and physicochemical properties, provides nature-inspired ideas leading to novel inhibitors of oncogenic protein kinases and related enzymes, capable to target a variety of cancer cells.

Structural and functional characterization of the triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 and its dimerization-dependent inhibitory activity

Phytocystatins are a group of proteins with significant potential to regulate activities of cysteine proteinases of native and pest/pathogen origins. The two-domain triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 was characterized in this study. This protein belongs to the second group of phytocystatins and contains all the conserved sequences and motifs as well as both N-terminal (CY) and C-terminal (CY-L) domains that are characteristic of phytocystatins with the C-terminal extension. We demonstrated that TrcC-8 forms stable dimers with a significantly reduced inhibitory activity against papain compared to the activity of monomers, indicating the regulatory nature of the oligomerization. Moreover, according to our research, only the N-terminal domain possesses the ability to form dimers, indicating that this part of TrcC-8 is involved in the dimerization of the full-length protein. Homology modelling of TrcC-8 strongly suggests distinct specificities for the CY and CY-L domains, confirmed in experiments with inhibition of the papain. Our results suggest that the CY domain of TrcC-8 may, although markedly weakly and suboptimally, interact with papain in an analogous mode to tarocystatin, while the CY-L domain of TrcC-8 has distinct specificity than tarocystatin.

Analogs of Cinnamic Acid Benzyl Amide As Nonclassical Inhibitors of Activated JAK2 Kinase

Scaffold-based analogs of cinnamic acid benzyl amide (CABA) exhibit pleiotropic effects in cancer cells, and their exact molecular mechanism of action is under investigation. The present study is part of our systemic analysis of interactions of CABA analogs with their molecular targets. These compounds were shown to inhibit Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and JAK2/signal transducer and activator of transcription 5 (STAT5) signaling and thus are attractive scaffolds for anticancer drug design. To identify the potential mechanisms of action of this class of compounds, direct interactions of the selected CABA analogs with JAK2 kinase were examined. Inhibition of JAK2 enzymatic activity was assessed, and molecular modeling studies of selected compounds-(E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide (WP1065), (E)-2-cyano-N-[(S)-1-phenylbutyl]- 3-(3-bromopyridin-2-yl)acrylamide (WP1130), and (E)-2-cyano-N-[(S)-1,4-diphenylbutyl]-3-(3-bromopyridin-2-yl)acrylamide (WP1702)-in the JAK2 kinase domain were used to support interpretation of the experimental data. Our results indicated that the tested CABA analogs are nonclassical inhibitors of activated (phosphorylated) JAK2, although markedly weaker than clinically tested ATP-competitive JAK2 inhibitors. Relatively small structural changes in the studied compounds affected interactions with JAK2, and their mode of action ranged from allosteric-noncompetitive to bisubstratecompetitive. These results demonstrated that direct inhibition of JAK2 enzymatic activity by the WP1065 (half-maximal inhibitory concentration [IC₅₀] = 14.8 µM), WP1130 (IC₅₀ = 3.8 µM), and WP1702 (IC₅₀ = 2.9 µM) potentially contributes, albeit minimally, to suppression of the JAK2/STAT signaling pathways in cancer cells and that additional specific structural modifications may amplify JAK2-inhibitory effects.