Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2.

Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M4S11 (α) and M3S9 (ß) clusters. Recent discoveries indicate that Zn(ii) ions are bound with MT2 with the range from nano- to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(ii)-depleted MT2 species. These forms serve as Zn(ii) donors or acceptors and are formed under varying cellular free Zn(ii) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(ii) binding mechanism in human MT2 with high resolution with respect to particular Zn(ii) binding sites, and provide structural insights into Zn(ii)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the α-domain is formed sequentially in the first stages, followed by the formation of the ß-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the ß-domain upon applying the seventh Zn(ii) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(ii) ion associates with the ß-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(ii) binding to the isolated ß-domain differs significantly from the whole protein, which explains its previously observed different Zn(ii)-binding properties. MD results obtained for Zn(ii) binding to the whole protein and isolated ß-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.

Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1.

In our previous work we demonstrated that a small protein called affibody can be used for a cytotoxic conjugate development. The anti-HER2 affibody was armed with one moiety of a highly potent auristatin E and specifically killed HER2-positive cancer cells with a nanomolar IC50. The aim of this study was to improve the anti-HER2 affibody conjugate by increasing its size and the number of conjugated auristatin molecules. The affibody was fused to the Fc fragment of IgG1 resulting in a dimeric construct with the molecular weight of 68 kDa, referred to as ZHER2:2891-Fc, ensuring its prolonged half-life in the blood. Due to the presence of four interchain cysteines, the fusion protein could carry four drug molecules. Notably, the in vitro tests of the improved anti-HER2 conjugate revealed that it exhibits the IC50 of 130 pM for the HER2-positive SK-BR-3 cells and 98 nM for the HER2-negative MDA-MB-231 cells. High efficacy and specificity of the auristatin conjugate based on ZHER2:2891-Fc indicate that this construct is suitable for further in vivo evaluation.

Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy.

Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody-drug conjugates. The FGF1V-valine-citrulline-MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V-vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality.

Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells.

The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2b-Fc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity.

Tumor-specific hyperthermia with aptamer-tagged superparamagnetic nanoparticles.

Targeted therapy is a method owing to its limited side effect profile, particularly in cancer treatment. Magnetic hyperthermia, which is induced by nanoparticles (NPs) conjugated with targeting agents, can be useful in combination with chemo- or radiotherapy. In this paper, we constructed dextran-coated ferric oxide NPs conjugated with specific anti-human epidermal growth factor receptor (HER2) aptamer and used them to induce magnetic hyperthermia in cultured cells. The specificity of the tagged NPs was determined by studying their effect relative to that of non-tagged NPs against two cell lines: human adenocarcinoma SK-BR3, overexpressing the HER2 receptor; and U-87 MG, a human glioblastoma epithelial cell line, not expressing HER2. In order to confirm the interaction of the tagged NPs with the cells we used, fluorescence microscopy and fluorescence-activated cell sorting analysis were performed. All of these experiments showed that the aptamer-tagged NPs were highly specific toward the HER2-expressing cells. In addition, a ninetyfold lower dose of the tagged NPs relative to that of the non-tagged NPs was needed to achieve ~50% cell killing by hyperthermia of the SK-BR3 cell line, while for the U-87 MG cells the viability level was close to 100%. These results show that targeted NPs can be applied at substantially lower doses than non-targeted ones to achieve similar effects of hyperthermia, which should greatly limit the side effects of treatment.

Protease resistant variants of FGF1 with prolonged biological activity.

Therapeutic potential of human acidic fibroblast growth factor (FGF1) resulting from its undeniable role in angiogenesis and wound healing processes is questioned due to its low stability and short half-life in vivo. Our previous studies showed that prolonged biological activity of FGF1 can be achieved by increasing its proteolytic resistance directly linked to improved global thermostability. In this study, we applied an alternative method of generation of long-lasting FGF1 variants by rigidification of the growth factor's segment highly sensitive to proteases action. In order to determine regions the most prone to enzymatic degradation, we used limited proteolysis by trypsin combined with mass spectrometry analysis. We found that the initial proteolytic cleavages occurred mainly within the C-terminal region of the wild-type protein, pointing on its significant role in growth factor degradation. Based on bioinformatic analysis, we introduced two single mutations (C117P, K118V) within ß-strand XI and combined them in a double mutant. We determined resistance to proteolysis, biophysical properties and biological activities of obtained variants. All of them occurred to be significantly less susceptible to trypsin (up to 100-fold) and also to chymotrypsin degradation comparing to the wild-type protein. Interestingly, all variants were not more thermostable than the wild-type FGF1. We attributed this dramatic increase in resistance to proteolysis to entropic stabilization of C-terminal region.

UDP-N-acetylglucosamine transporter (SLC35A3) regulates biosynthesis of highly branched N-glycans and keratan sulfate.

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply ß-1,3-N-acetylglucosaminyl-transferase 7 (ß3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein ß-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.

FGF1-gold nanoparticle conjugates targeting FGFR efficiently decrease cell viability upon NIR irradiation.

Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy.

Characterization of [4Fe-4S]-containing and cluster-free forms of Streptomyces WhiD.

WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster, and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from Escherichia coli as a soluble protein containing a single [4Fe-4S](2+) cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and pH 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance. [4Fe-4S](2+) WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apoprotein destabilized the protein fold significantly but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay, demonstrating that it does not function as a general protein disulfide reductase.

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): purification, primary structure, and inhibitory properties.

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K(a)=1.1 x 10(9) M(-1) and 2.5 x 10(9) M(-1), respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K(a)=4.5 x 10(8) M(-1) and 1.2 x 10(10) M(-1)). Weak interaction with human plasmin (K(a)=1.2 x 10(7) M(-1)) was also revealed.

Assignment of the zinc ligands in RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor.

ZAS proteins are widespread bacterial zinc-containing anti-sigma factors that regulate the activity of sigma factors in response to diverse cues. One of the best characterized ZAS proteins is RsrA from Streptomyces coelicolor, which responds to disulfide stress. Zn-RsrA binds and represses the transcriptional activity of sigmaR in the reducing environment of the cytoplasm but undergoes reversible, intramolecular disulfide bond formation during oxidative stress. This expels the single metal ion and causes dramatic structural changes in RsrA that result in its dissociation from sigmaR, leaving the sigma factor free to activate the transcription of antioxidant genes. We showed recently that Zn2+ serves a critical role in modulating the redox activity of RsrA thiols but uncertainty remains as to how the metal ion is coordinated in RsrA and related ZAS proteins. Using a combination of random and site-specific mutagenesis with zinc K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy, we have assigned unambiguously the metal ligands in RsrA, thereby distinguishing between the different ligation models that have been proposed. The data show that the zinc site in RsrA is comprised of Cys11, His37, Cys41, and Cys44. Three of these residues are part of a conserved ZAS-specific sequence motif (H37xxxC41xxC44), with the fourth ligand, Cys11, found in a subset of ZAS proteins. Cys11 and Cys44 form the trigger disulfide in RsrA, explaining why the metal ion is expelled during oxidation. We discuss these data in the context of redox sensing by RsrA and the sensory mechanisms of other ZAS proteins.

Evidence that the Streptomyces developmental protein WhiD, a member of the WhiB family, binds a [4Fe-4S] cluster.

WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained approximately 4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g approximately 2.06, 1.94) consistent with a one-electron reduction of a [4Fe-4S](2+) cluster to a [4Fe-4S](1+) state with electron spin of S = (1/2). The anaerobically reconstituted [4Fe-4S] cluster was oxygen sensitive. Upon exposure to air, absorption at 410 and 505 nm first increased and then showed a steady decrease with time until the protein was colorless in the near UV/visible region. These changes are consistent with an oxygen-induced change from a [4Fe-4S] to a [2Fe-2S] cluster, followed by complete loss of cluster from the protein. Each of the four conserved cysteine residues, Cys-23, -53, -56, and -62, was essential for WhiD function in vivo.