Dmitri Tolkatchev

Structure dissection of human progranulin identifies well-folded granulin/epithelin modules with unique functional activities.

Progranulin is a secreted protein with important functions in several physiological and pathological processes, such as embryonic development, host defense, and wound repair. Autosomal dominant mutations in the progranulin gene cause frontotemporal dementia, while overexpression of progranulin promotes the invasive progression of a range of tumors, including those of the breast and the brain. Structurally, progranulin consists of seven‐and‐a‐half tandem repeats of the granulin/epithelin module (GEM), several of which have been isolated as discrete 6‐kDa GEM peptides. We have expressed all seven human GEMs using recombinant DNA in Escherichia coli. High‐resolution NMR showed that only the three GEMs, hGrnA, hGrnC, and hGrnF, contain relatively well‐defined three‐dimensional structures in solution, while others are mainly mixtures of poorly structured disulfide isomers. The three‐dimensional structures of hGrnA, hGrnC, and hGrnF contain a stable stack of two β‐hairpins in their N‐terminal subdomains, but showed a more flexible C‐terminal subdomain. Interestingly, of the well‐structured GEMs, hGrnA demonstrated potent growth inhibition of a breast cancer cell line, while hGrnF was stimulatory. Poorly folded peptides were either weakly inhibitory or without activity. The functionally active and structurally well‐characterized human hGrnA offers a unique opportunity for detailed structure–function studies of these important GEM proteins as novel members of mammalian growth factors.

Three-dimensional structure and ligand interactions of the low molecular weight protein tyrosine phosphatase from Campylobacter jejuni

A putative low molecular weight protein tyrosine phosphatase (LMW‐PTP) was identified in the genome sequence of the bacterial pathogen, Campylobacter jejuni. This novel gene, cj1258, has sequence homology with a distinctive class of phosphatases widely distributed among prokaryotes and eukaryotes. We report here the solution structure of Cj1258 established by high‐resolution NMR spectroscopy using NOE‐derived distance restraints, hydrogen bond data, and torsion angle restraints. The three‐dimensional structure consists of a central four‐stranded parallel β‐sheet flanked by five α‐helices, revealing an overall structural topology similar to those of the eukaryotic LMW‐PTPs, such as human HCPTP‐A, bovine BPTP, and Saccharomyces cerevisiae LTP1, and to those of the bacterial LMW‐PTPs MPtpA from Mycobacterium tuberculosis and YwlE from Bacillus subtilis. The active site of the enzyme is flexible in solution and readily adapts to the binding of ligands, such as the phosphate ion. An NMR‐based screen was carried out against a number of potential inhibitors and activators, including phosphonomethylphenylalanine, derivatives of the cinnamic acid, 2‐hydroxy‐5‐nitrobenzaldehyde, cinnamaldehyde, adenine, and hypoxanthine. Despite its bacterial origin, both the three‐dimensional structure and ligand‐binding properties of Cj1258 suggest that this novel phosphatase may have functional roles close to those of eukaryotic and mammalian tyrosine phosphatases. The three‐dimensional structure along with mapping of small‐molecule binding will be discussed in the context of developing high‐affinity inhibitors of this novel LMW‐PTP.

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenins propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.

Molecular imaging of breast tumors using a near‐infrared fluorescently labeled clusterin binding peptide

Several reports have shown that secreted clusterin (sCLU) plays multiple roles in tumor development and metastasis. Here, we report on a 12‐mer sCLU binding peptide (designated P3378) that was identified by screening a phage‐display peptide library against purified human sCLU. Differential resonance perturbation nuclear magnetic resonance using P3378 and a scrambled control peptide (designated P3378R) confirmed the P3378‐sCLU interaction and demonstrated that it was sequence specific. P3378 and P3378R peptides were conjugated to an Alexa680 near infrared fluorophore (NIRF) and assessed for their tumor homing abilities in in vivo time‐domain fluorescence optical imaging experiments using living 4T1 tumor bearing BALB/c mice. When injected in separate animals, both peptides accumulated at the tumor site, however the NIRF‐labeled P3378 peptide was retained for a significant longer period of time than the P3378R peptide. Similar observations were made after simultaneously injecting the same tumor‐bearing animal with a peptide mixture of P3378 DyLight (DL)680 and the P3378R‐DL800. Coinjection of P3378‐DL680 with excess unlabeled P3378 blocked tumor accumulation of fluorescent signal while excess P3378R control peptide did not confirming the sequence specificity of the tumor accumulation. Finally, ex vivo fluorescence microscopy of these tumors confirmed the presence of P3378‐DL680 in the tumor and its colocalization with CLU. These results confirm the tumor targeting specificity of the P3378 CLU‐binding peptide and suggest its usefulness for the in vivo monitoring of solid tumors secreting detectable levels of CLU.

Piperine, an alkaloid inhibiting the super-relaxed state of myosin, binds to the myosin regulatory light chain

Piperine, an alkaloid from black pepper, was found to inhibit the super-relaxed state (SRX) of myosin in fast-twitch skeletal muscle fibers. In this work we report that the piperine molecule binds heavy meromyosin (HMM), whereas it does not interact with the regulatory light chain (RLC)-free subfragment-1 (S1) or with control proteins from the same muscle molecular machinery, G-actin and tropomyosin. To further narrow down the location of piperine binding, we studied interactions between piperine and a fragment of skeletal myosin consisting of the full-length RLC and a fragment of the heavy chain (HCF). The sequence of HCF was designed to bind RLC and to dimerize via formation of a stable coiled coil, thus producing a well-folded isolated fragment of the myosin neck. Both chains were co-expressed in Escherichia coli, the RLC/HCF complex was purified and tested for stability, composition and binding to piperine. RLC and HCF chains formed a stable heterotetrameric complex (RLC/HCF)2 which was found to bind piperine. The piperine molecule was also found to bind isolated RLC. Piperine binding to RLC in (RLC/HCF)2 altered the compactness of the complex, suggesting that the mechanism of SRX inhibition by piperine is based on changing conformation of the myosin.