Check out our New Publishers' Select for Free Articles

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.41, #5)

Protein and peptide probes for molecular imaging by Xiaoyuan Chen (pp. 1009-1012).

Protein-based tumor molecular imaging probes by Xin Lin; Jin Xie; Xiaoyuan Chen (pp. 1013-1036).

Molecular imaging is an emerging discipline which plays critical roles in diagnosis and therapeutics. It visualizes and quantifies markers that are aberrantly expressed during the disease origin and development. Protein molecules remain to be one major class of imaging probes, and the option has been widely diversified due to the recent advances in protein engineering techniques. Antibodies are part of the immunosystem which interact with target antigens with high specificity and affinity. They have long been investigated as imaging probes and were coupled with imaging motifs such as radioisotopes for that purpose. However, the relatively large size of antibodies leads to a half-life that is too long for common imaging purposes. Besides, it may also cause a poor tissue penetration rate and thus compromise some medical applications. It is under this context that various engineered protein probes, essentially antibody fragments, protein scaffolds, and natural ligands have been developed. Compared to intact antibodies, they possess more compact size, shorter clearance time, and better tumor penetration. One major challenge of using protein probes in molecular imaging is the affected biological activity resulted from random labeling. Site-specific modification, however, allows conjugation happening in a stoichiometric fashion with little perturbation of protein activity. The present review will discuss protein-based probes with focus on their application and related site-specific conjugation strategies in tumor imaging.

Keywords: Tumor targeting; Antibody; Human epidermal growth factor receptor (HER); Vascular endothelial growth factor (VEGF)

Protein scaffold-based molecular probes for cancer molecular imaging by Zheng Miao; Jelena Levi; Zhen Cheng (pp. 1037-1047).

Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.

Keywords: Protein scaffolds; Peptide, molecular imaging; Cancer; PET

Radiolabeled CCK/gastrin peptides for imaging and therapy of CCK2 receptor-expressing tumors by Susan Roosenburg; Peter Laverman; Floris L. van Delft; Otto C. Boerman (pp. 1049-1058).

Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, like medullary thyroid carcinomas, small cell lung cancers and stromal ovarian cancers. The specific receptor-binding property of the endogenous ligands for these receptors can be exploited by labeling peptides with a radionuclide and using these as carriers to guide the radioactivity to the tissues that express the receptors. In this way, tumors can be visualized using positron emission tomography and single photon emission computed tomography imaging. A variety of radiolabeled CCK/gastrin-related peptides has been synthesized and characterized for imaging. All peptides have the C-terminal CCK receptor-binding tetrapeptide sequence Trp-Met-Asp-Phe-NH₂ in common or derivatives thereof. This review focuses on the development and application of radiolabeled CCK/gastrin peptides for radionuclide imaging and radionuclide therapy of tumors expressing CCK receptors. We discuss both preclinical studies as well as clinical studies with CCK and gastrin peptides.

Keywords: Cholecystokinin-2 receptors; Peptides; Minigastrin; CCK8; Medullary thyroid carcinoma

Impact of bifunctional chelators on biological properties of ¹¹¹In-labeled cyclic peptide RGD dimers by Jiyun Shi; Young-Seung Kim; Sudipta Chakraborty; Yang Zhou; Fan Wang; Shuang Liu (pp. 1059-1070).

The present study describes the synthesis and biological evaluation of ¹¹¹In(DOTA-3P-RGD₂) (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid; 3P-RGD₂ = PEG₄-E[PEG₄-c(RGDfK)]₂; PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), ¹¹¹In(DTPA-3P-RGD₂) (DTPA = diethylenetriaminepentaacetic acid) and ¹¹¹In(DTPA-Bn-3P-RGD₂) (DTPA-Bn = 2-(p-thioureidobenzyl)-diethylenetriaminepentaacetic acid) as potential radiotracers for imaging tumor integrin α_vβ₃ expression in athymic nude mice bearing U87MG glioma xenografts. The aim of the study is to assess the impact of the bifunctional chelator (BFC) (DOTA vs. DTPA or DTPA-Bn) on the biodistribution characteristics of the ¹¹¹In-labeled 3P-RGD₂. IC₅₀ values of DOTA-3P-RGD₂, DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂ were determined to be 1.3 ± 0.2, 1.4 ± 0.3, 1.3 ± 0.3 nM, respectively, against ¹²⁵I-c(RGDyK) bound to U87MG human glioma cells. Radiotracers were prepared by reacting ¹¹¹InCl₃ with the RGD peptide conjugates in NH₄OAc buffer (100 mM, pH 5.5). For DOTA-3P-RGD₂, successful radiolabeling could be completed by heating the reaction mixture at 100°C for 15–20 min. For DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂, the radiolabeling was almost instantaneous at room temperature. The specific activity was ~50 mCi/mg (or ~100 mCi/μmol) for ¹¹¹In(DOTA-3P-RGD₂) and ~200 mCi/mg (or ~400 mCi/μmol) for ¹¹¹In(DTPA-3P-RGD₂). The results from biodistribution studies showed that all the three radiotracers have high tumor uptake and excellent tumor-to-background (T/B) ratios up to 4-h postinjection. After that time point, both ¹¹¹In(DTPA-3P-RGD₂) and ¹¹¹In(DTPA-Bn-3P-RGD₂) showed a much faster tumor washout and poorer T/B ratios than ¹¹¹In(DOTA-3P-RGD₂). The tumor uptake of ¹¹¹In(DOTA-3P-RGD₂) is integrin α_vβ₃- and RGD-specific. ¹¹¹In(DOTA-3P-RGD₂) is metabolically stable while only ~25% of ¹¹¹In(DTPA-Bn-3P-RGD₂) remains intact in the feces during 2-h period. On the basis of results from this study, it was concluded that ¹¹¹In(DTPA-3P-RGD₂) can be an effective integrin α_vβ₃-targeted radiotracer if the high-specific activity is required. However, DOTA remains to be the BFC of choice for the development of therapeutic lanthanide radiotracers.

Keywords: Integrin α_vβ₃ ; ¹¹¹In-labeled cyclic RGD peptides; Tumor imaging

Lantibiotics as probes for phosphatidylethanolamine by Ming Zhao (pp. 1071-1079).

Phosphatidylethanolamine (PE) is a major component in the mammalian plasma membrane. It is present mainly in the inner leaflet of the membrane bilayer in a viable, typical mammalian cell. However, accumulating evidence indicates that a number of biological events involve PE externalization. For instance, PE is concentrated at the surface of cleavage furrow between mitotic daughter cells and is correlated with the dynamics of contractile ring. In apoptotic cells, PE is exposed to the cell surface, thus providing a molecular marker for detection. In addition, PE is a cofactor in the anticoagulant mechanism, and a distinct distribution profile of PE has been documented at the blood–endothelium interface. These recent discoveries were made possible using PE-specific probes derived from duramycin and cinnamycin, which are members of type B lantibiotics. This review provides an account on the features of these PE-specific lantibiotics in the context of molecular probes for the characterization of PE on a cellular and tissue level. According to the existing data, PE is likely a versatile chemical species that plays a role in the regulation of defined biological and physiological activities. The utilities of lantibiotic-based molecular probes will help accelerate the characterization of PE as an abundant, yet elusive membrane component.

Keywords: Duramycin; Cinnamycin; Phosphatidylethanolamine; Anticoagulant; Apoptosis; Cytokinesis; Imaging probe

Peptide heterodimers for molecular imaging by Yongjun Yan; Xiaoyuan Chen (pp. 1081-1092).

One main issue with peptide-based molecular imaging probes is their relatively low tumor affinity and short retention time. To improve peptide binding affinity, multivalency approach has been introduced. Traditionally, this approach involves the use of peptide homodimers or homomultimers in which peptide ligands of the same type are constructed with suitable linkers. Recently, a new approach using peptide heterodimers has emerged as a promising method for targeting multi-receptor over-expressed tumor cells. Significant affinity enhancements have been observed with peptide heterodimers compared with their parent peptide monomers. In a peptide heterodimer, two different peptide ligands capable of targeting two different receptors are covalently linked. The binding modes of peptide heterodimers can be monovalent or bivalent depending on whether simultaneous binding of two ligands can be achieved. Increased local ligand concentration and improved binding kinetics contribute to enhanced binding in both monovalent- and bivalent binding modes, while multivalency effect also plays an important role in bivalent binding mode. As many tumors overexpress multiple receptors, more peptide heterodimer-based molecular imaging probes are expected to be developed in future. This review article will discuss the peptide homodimers and heterodimers for molecular imaging with special emphasis on peptide heterodimers.

Keywords: Peptide; Heterodimer; Molecular imaging

A cell permeable peptide analog as a potential-specific PET imaging probe for prostate cancer detection by Guiyang Hao; Jian Zhou; Yi Guo; Michael A. Long; Tiffani Anthony; Jennifer Stanfield; Jer-Tsong Hsieh; Xiankai Sun (pp. 1093-1101).

Non-invasive detection of prostate cancer or metastases still remains a challenge in the field of molecular imaging. In our recent work of screening arginine- or lysine-rich peptides for intracellular delivery of a therapeutic agent into prostate cancer cells, an arginine-rich cell permeable peptide (NH₂GR₁₁) was found with an unexpectedly preferential uptake in prostate cancer cell lines. The goal of this work was to develop this peptide as a positron emission tomography (PET) imaging probe for specific detection of distant prostate cancer metastases. The optimal length of arginine-rich peptides was evaluated by the cell uptake efficiency of three fluorescein isothiocyanate (FITC)-tagged oligoarginines (NHGR₉, NHGR₁₁, and NHGR₁₃) in four human prostate cell lines (LNCaP, PZ-HPV-7, DU145, and PC3). Of the three oligoarginines, NH₂GR₁₁ showed the highest cell uptake and internalization efficiency with its subcellular localization in cytosol. The biodistribution of FITC-NHGR₉, FITC-NHGR₁₁, and FITC-NHGR₁₃ performed in control nude mice displayed the unique preferential accumulation of FITC-NHGR₁₁ in the prostate tissue. Further in vivo evaluation of FITC-NHGR₁₁ in PC3 tumor-bearing nude mice revealed elevated uptake of this peptide in tumors as compared to other organs. In vivo pharmacokinetics evaluated with ⁶⁴Cu-labeled NH₂GR₁₁ showed that the peptide was rapidly cleared from the blood (t _1/2 = 10.7 min) and its elimination half-life was 17.2 h. The PET imaging specificity of ⁶⁴Cu-labled NH₂GR₁₁ was demonstrated for the detection of prostate cancer in a comparative imaging experiment using two different human cancer xenograft models.

Keywords: PET; Prostate cancer; Cell permeable peptide; ⁶⁴Cu

Phage display peptide probes for imaging early response to bevacizumab treatment by Qizhen Cao; Shuanglong Liu; Gang Niu; Kai Chen; Yongjun Yan; Zhaofei Liu; Xiaoyuan Chen (pp. 1103-1112).

Early evaluation of cancer response to a therapeutic regimen can help increase the effectiveness of treatment schemes and, by enabling early termination of ineffective treatments, minimize toxicity, and reduce expenses. Biomarkers that provide early indication of tumor therapy response are urgently needed. Solid tumors require blood vessels for growth, and new anti-angiogenic agents can act by preventing the development of a suitable blood supply to sustain tumor growth. The purpose of this study is to develop a class of novel molecular imaging probes that will predict tumor early response to an anti-angiogenic regimen with the humanized vascular endothelial growth factor antibody bevacizumab. Using a bevacizumab-sensitive LS174T colorectal cancer model and a 12-mer bacteriophage (phage) display peptide library, a bevacizumab-responsive peptide (BRP) was identified after six rounds of biopanning and tested in vitro and in vivo. This 12-mer peptide was metabolically stable and had low toxicity to both endothelial cells and tumor cells. Near-infrared dye IRDye800-labeled BRP phage showed strong binding to bevacizumab-treated tumors, but not to untreated control LS174T tumors. In addition, both IRDye800- and ¹⁸F-labeled BRP peptide had significantly higher uptake in tumors treated with bevacizumab than in controls treated with phosphate-buffered saline. Ex vivo histopathology confirmed the specificity of the BRP peptide to bevacizumab-treated tumor vasculature. In summary, a novel 12-mer peptide BRP selected using phage display techniques allowed non-invasive visualization of early responses to anti-angiogenic treatment. Suitably labeled BRP peptide may be potentially useful pre-clinically and clinically for monitoring treatment response.

Keywords: Phage display; Angiogenesis; Therapy response; Bevacizumab; Molecular imaging

Optimization of matrix metalloproteinase fluorogenic probes for osteoarthritis imaging by Ju Hee Ryu; Aeju Lee; Jin Hee Na; Seulki Lee; Hyung Jun Ahn; Jong Woong Park; Cheol-Hee Ahn; Byung-Soo Kim; Ick Chan Kwon; Kuiwon Choi; Inchan Youn; Kwangmeyung Kim (pp. 1113-1122).

Among the classical collagenases, matrix metalloproteinase-13 (called MMP-13, collagenase-3) is one of the most important components for cartilage destruction of osteoarthritis (OA) developments. Despite many efforts, the detection methods of MMP-13 activity have been met with limited success in vivo, in part, due to the low sensitivity and low selectivity by homology of MMP family. Previously, we demonstrated the use of strongly dark-quenched fluorogenic probe allowed for the visual detection of MMP-13 in vitro and in OA-induced rat models. In this study, we described the optimization of MMP-13 fluorogenic probe for OA detection in vivo. Three candidate probes demonstrated recovered fluorescent intensity proportional with MMP-13 concentrations, respectively; however, Probe 2 exhibited both high signal amplification and selective recognition for MMP-13, not MMP-2 and MMP-9 in vitro. When Probe 2 was applied to OA-induced rat models, clear visualization of MMP-13 activity in OA-induced cartilage was obtained. Optimized MMP-13 fluorogenic probe can be applied to detect and image OA and have potential for evaluating the in vivo efficacy of MMP-13 inhibitors which are being tested for therapeutic treatment of OA.

Keywords: Matrix metalloproteinase (MMP); Osteoarthritis; Fluorogenic probe; Optical imaging; Peptide

Peptide-based molecular beacons for cancer imaging and therapy by Tracy W. B. Liu; Juan Chen; Gang Zheng (pp. 1123-1134).

Peptide-based molecular beacons are Förster resonance energy transfer-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets and thus are useful tools for in vivo cancer imaging. With our increasing knowledge about human genome in health and disease, peptide-based “smart” probes are continually developed for in vivo optical imaging of specific molecular targets, biological pathways and cancer progression and diagnosis. A class of fluorescent photosensitizers further extends the application of peptide beacons to cancer therapeutics. This review highlights the applications of peptide beacons in cancer imaging, the simultaneous treatment and response monitoring and smart therapeutics with a focus on recent improvements in the design of these probes.

Keywords: Molecular beacons; Optical imaging; Photodynamic therapy; Activatable probes; Proteases

Amino acids special issue ‘Protein interactions in the virus–host relationship’ by Alessandro Marcello (pp. 1135-1136).

Identification of cellular factors binding to acetylated HIV-1 integrase by Awatef Allouch; Anna Cereseto (pp. 1137-1145).

The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070–3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein–protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291–297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle.

Keywords: HIV-1 integrase; Histones acetyltransferase; Acetylation; Tethered catalysis system; Yeast two-hybrid screening; Integrase binding factors

Virus entry paradigms by Manjula Kalia; Shahid Jameel (pp. 1147-1157).

Viruses, despite being relatively simple in structure and composition, have evolved to exploit complex cellular processes for their replication in the host cell. After binding to their specific receptor on the cell surface, viruses (or viral genomes) have to enter cells to initiate a productive infection. Though the entry processes of many enveloped viruses is well understood, that of most non-enveloped viruses still remains unresolved. Recent studies have shown that compared to direct fusion at the plasma membrane, endocytosis is more often the preferred means of entry into the target cell. Receptor-mediated endocytic pathways such as the dynamin-dependent clathrin and caveolar pathways are well characterized as viral entry portals. However, many viruses are able to utilize multiple uptake pathways. Fluid phase uptake, though relatively non-specific in terms of its cargo, potentially aids viral infection by its ability to intersect with the endocytic pathway. In fact, many viruses despite using specialized pathways for entry are still able to generate productive infection via fluid phase uptake. Macropinocytosis, a major fluid uptake pathway found in epithelial cells and fibroblasts, is stimulated by growth factor receptors. Many viruses can induce these signaling cascades in cells leading to macropinocytosis. Though endocytic trafficking is utilized by both enveloped and non-enveloped viruses, key differences lie in the way membranes are traversed to deposit the viral genome at its site of replication. This review will discuss recent developments in the rapidly evolving field of viral entry.

Keywords: Enveloped virus; Non-enveloped virus; Endocytosis; Membrane fusion; Signaling

Molecular mechanisms of flavivirus membrane fusion by Karin Stiasny; Richard Fritz; Karen Pangerl; Franz X. Heinz (pp. 1159-1163).

Flaviviruses comprise a number of important human pathogens including yellow fever, dengue, West Nile, Japanese encephalitis and tick-borne encephalitis viruses. They are small enveloped viruses that enter cells by receptor-mediated endocytosis and release their nucleocapsid into the cytoplasm by fusing their membrane with the endosomal membrane. The fusion event is triggered by the acidic pH in the endosome and is mediated by the major envelope protein E. Based on the atomic structures of the pre- and post-fusion conformations of E, a fusion model has been proposed that includes several steps leading from the metastable assembly of E at the virion surface to membrane merger and fusion pore formation trough conversion of E into a stable trimeric post-fusion conformation. Using recombinant subviral particles of tick-borne encephalitis virus as a model, we have defined individual steps of the molecular processes underlying the flavivirus fusion mechanisms. This includes the identification of a conserved histidine as being part of the pH sensor in the fusion protein that responds to the acidic pH and thus initiates the structural transitions driving fusion.

Keywords: Flavivirus; Membrane fusion; Viral fusion protein; Fusion trigger; Histidine

Importance of exercise immunology in health promotion by J. C. Rosa Neto; F. S. Lira; M. T. de Mello; Ronaldo Vagner T. Santos (pp. 1165-1172).

Chronic physical exercise with adequate intensity and volume associated with sufficient recovery promotes adaptations in several physiological systems. While intense and exhaustive exercise is considered an important immunosuppressor agent and increases the incidence of upper respiratory tract infections (URTI), moderate regular exercise has been associated with significant disease protection and is a complementary treatment of many chronic diseases. The effects of chronic exercise occur because physical training can induce several physiological, biochemical and psychological adaptations. More recently, the effect of acute exercise and training on the immunological system has been discussed, and many studies suggest the importance of the immune system in prevention and partial recovery in pathophysiological situations. Currently, there are two important hypotheses that may explain the effects of exercise and training on the immune system. These hypotheses including (1) the effect of exercise upon hormones and cytokines (2) because exercise can modulate glutamine concentration. In this review, we discuss the hypothesis that exercise may modulate immune functions and the importance of exercise immunology in respect to chronic illnesses, chronic heart failure, malnutrition and inflammation.

Keywords: Acute physical exercise; Chronic physical exercise; Moderate training; Glutamine; Inflammation; Immunosuppression

Kynurenine metabolism in health and disease by Lukasz R. Kolodziej; Ewa M. Paleolog; Richard O. Williams (pp. 1173-1183).

Kynurenine is a small molecule derived from tryptophan when this amino acid is metabolised via the kynurenine pathway. The biological activity of kynurenine and its metabolites (kynurenines) is well recognised. Therefore, understanding the regulation of the subsequent biochemical reactions is essential for the design of therapeutic strategies which aim to interfere with the kynurenine pathway. However, kynurenine concentration in the body may not only be determined by the efficiency of kynurenine synthesis but also by the rate of kynurenine clearance. In this review, current knowledge about the mechanisms of kynurenine production and routes of its clearance is presented. In addition, the involvement of kynurenine and its metabolites in the biology of different T cell subsets (including Th17 cells and regulatory T cells) and neuronal cells is discussed.

Keywords: Brain; Indoleamine 2,3 dioxygenase; Immunity; Kynurenine; Th17; Treg

Leucine nutrition in animals and humans: mTOR signaling and beyond by Fengna Li; Yulong Yin; Bie Tan; Xiangfeng Kong; Guoyao Wu (pp. 1185-1193).

Macronutrients, such as protein or amino acid, not only supply calories but some components may also play as signaling molecules to affect feeding behavior, energy balance, and fuel efficiency. Leucine, a branched-chain amino acid is a good example. After structural roles are satisfied, the ability of leucine to function as signal and oxidative substrate is based on a sufficient intracellular concentration. Therefore, leucine level must be sufficiently high to play the signaling and metabolic roles. Leucine is not only a substrate for protein synthesis of skeletal muscle, but also plays more roles beyond that. Leucine activates signaling factor of mammalian target of rapamycin (mTOR) to promote protein synthesis in skeletal muscle and in adipose tissue. It is also a major regulator of the mTOR sensitive response of food intake to high protein diet. Meanwhile, leucine regulates blood glucose level by promoting gluconeogenesis and aids in the retention of lean mass in a hypocaloric state. It is beneficial to animal nutrition and clinical application and extrapolation to humans.

Keywords: Leucine nutrition; mTOR signaling

Tryptophan metabolism, from nutrition to potential therapeutic applications by Nathalie Le Floc’h; Winfried Otten; Elodie Merlot (pp. 1195-1205).

Tryptophan is an indispensable amino acid that should to be supplied by dietary protein. Apart from its incorporation into body proteins, tryptophan is the precursor for serotonin, an important neuromediator, and for kynurenine, an intermediary metabolite of a complex metabolic pathway ending with niacin, CO₂, and kynurenic and xanthurenic acids. Tryptophan metabolism within different tissues is associated with numerous physiological functions. The liver regulates tryptophan homeostasis through degrading tryptophan in excess. Tryptophan degradation into kynurenine by immune cells plays a crucial role in the regulation of immune response during infections, inflammations and pregnancy. Serotonin is synthesized from tryptophan in the gut and also in the brain, where tryptophan availability is known to influence the sensitivity to mood disorders. In the present review, we discuss the major functions of tryptophan and its role in the regulation of growth, mood, behavior and immune responses with regard to the low availability of this amino acid and the competition between tissues and metabolic pathways for tryptophan utilization.

Keywords: Tryptophan; Metabolism; Nutrition; Immune response; Mood disorders

Synergic effect of α-tocopherol and naringenin in transglutaminase-induced differentiation of human prostate cancer cells by Piera Torricelli; Pasquale Ricci; Bruno Provenzano; Alessandro Lentini; Claudio Tabolacci (pp. 1207-1214).

Prostate cancer is the second most common cancer in men worldwide. Its prevention and treatment remain a challenge to clinicians. Thus, there is an urgent need to discover novel, less toxic, and more effective therapies for patients. Many vitamins and related chemicals, including vitamin E, (tocopherols) have shown their anti-cancer activities as anti-oxidants, activators of transcription factors or factors influencing epigenetic events. Although laboratory tests including the use of animal models showed that this vitamin may have anticancer properties, whether it can effectively prevent the development and/or progression of prostate cancer in humans remains to be intensively studied. This review provides up-to-date information regarding the recent outcomes of laboratory, epidemiology and/or clinical trials on the effects of tocopherols on prostate cancer development, along with our last observations on a combined treatment of a prostate cancer cell line (PC-3) with two natural antineoplastic compounds, naringenin (NG) and α-tocopherol (α-TOC). We report the synergic effect of α-TOC and NG in transglutaminase-induced differentiation of human PC-3 prostate cancer cells. While our results are based on one histological class of tumor, the most significant implication of this observation is that establishes a new way in the screening for detecting new differentiative antineoplastic agents.

Keywords: α-Tocopherol; Naringenin; Transglutaminase; Differentiation; Apoptosis; Human PC-3 prostate cancer cell

Synthesis of biodegradable chiral poly(ester-imide)s derived from valine-, leucine- and tyrosine-containing monomers by Shadpour Mallakpour; Parvin Asadi; Mohammad R. Sabzalian (pp. 1215-1222).

The present demand for a drastic reduction in environmental pollution is extended to qualitative change in the approach to development of biodegradable polymers. The aim of this article is to focus on the synthesis of biodegradable optically active poly(ester-imide)s (PEI)s, which compose of different amino acids in the main chain as well as in the side chain. These polymers were synthesized by polycondensation of diacid monomers such as 5-(2-phthalimidyl-3-methyl butanoylamino) isophthalic acid (1), 5-(4-methyl-2-phthalimidyl pentanoylamino)isophthalic acid (2) with N,N′-(pyromellitoyl)-bis-l-tyrosine dimethyl ester (3) as a phenolic diol. The direct polycondensation reaction was carried out in a system of tosyl chloride, pyridine and N,N-dimethylformamide as a condensing agent under conventional heating conditions. The optically active PEIs were obtained in good yield and moderate inherent viscosity. The synthesized polymers were characterized by means of FT-IR, ¹H-NMR, elemental and thermo gravimetric analysis techniques. In addition, in vitro toxicity and soil burial test were employed for assessing the sensitivity of these compounds to microbial degradation. To this purpose, biodegradability behavior of the monomers and polymers were investigated in culture media and soil condition. The results of this study revealed that synthesized monomers and their derived polymers are biologically active and probably microbiologically biodegradable.

Keywords: Biodegradable polymers; Polymers containing amino acid; Optically active material; Poly(ester-imide)

Omentin inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells through the PI3K/Akt pathway by Xin-Yun Duan; Ping-Li Xie; Yu-Lin Ma; Si-Yuan Tang (pp. 1223-1231).

Arterial calcification is positively associated with visceral adiposity, but the mechanisms remain unclear. Omentin is a novel adipokine that is selectively expressed in visceral adipose tissue. The levels of circulating omentin are decreased in obesity, and they correlate negatively with waist circumference. This study investigated the effects of omentin on the osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Omentin inhibited mRNA expression of alkaline phosphatase (ALP) and osteocalcin; omentin also suppressed ALP activity, osteocalcin protein production, and the matrix mineralization. Furthermore, omentin selectively activated phosphatidylinositol 3-kinase (PI3K) downstream effector Akt. Moreover, inhibition of PI3K or Akt activation reversed the effects of omentin on ALP activity and the matrix mineralization. The present results demonstrate for the first time that omentin can inhibit osteoblastic differentiation of CVSMCs via PI3K/Akt signaling pathway, suggesting that the lower omentin levels in obese (specially visceral obese) subjects contribute to the development of arterial calcification, and omentin plays a protective role against arterial calcification.

Keywords: Omentin; Vascular smooth muscle cells; Calcification; Phosphatidylinositol 3-kinase

Role of low native state kinetic stability and interaction of partially unfolded states with molecular chaperones in the mitochondrial protein mistargeting associated with primary hyperoxaluria by Angel L. Pey; Eduardo Salido; Jose M. Sanchez-Ruiz (pp. 1233-1245).

The G170R variant of the alanine:glyoxylate aminotransferase (AGT) is the most common pathogenic allele associated to primary hyperoxaluria type I (PH1), leading to mitochondrial mistargeting when combined with the P11L and I340M polymorphisms (minor allele; AGT_LM). In this work, we have performed a comparative analysis on the conformation, unfolding energetics and interaction with molecular chaperones between AGT_wt, AGT_LM and AGT_LRM (G170R in the minor allele) proteins. Our results show that these three variants share similar conformational and functional properties as folded dimers. However, kinetic stability analyses showed a ≈1,000-fold increased unfolding rate for apo-AGT_LRM compared to apo-AGT_wt, as well as a reduced folding efficiency upon expression in Escherichia coli. Pyridoxal 5′-phosphate (PLP)-binding provided a 4–5 orders of magnitude enhancement of the kinetic stability for all variants, suggesting a role for kinetic stabilization in pyridoxine-responsive PH1. Conformational studies at mild acidic pH and moderate guanidium concentrations showed the formation of a molten-globule-like unfolding intermediate in all three variants, which do not reactivate to the native state and strongly interact with Hsc70 and Hsp90 chaperones. Additional expression analyses in a mammalian cell-free system at neutral pH showed enhanced interaction of AGT_LRM with Hsc70 and Hsp90 proteins compared to AGT_wt, suggesting kinetic trapping of the mutant by chaperones along the folding process. Overall, our results suggest that mitochondrial mistargeting of AGT_LRM may involve the presentation of AGT partially folded states to the mitochondrial import machinery by molecular chaperones, which would be facilitated by the low native state kinetic stability (partially corrected by PLP binding) and kinetic trapping during folding of the AGT_LRM variant with molecular chaperones.

Keywords: Alanine:glyoxylate aminotransferase; Protein kinetic stability; Partially folded states; Molecular chaperones; Primary hyperoxaluria type 1

Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase by Vitalia V. Koulikova; Lyudmila N. Zakomirdina; Olga I. Gogoleva; Marina A. Tsvetikova; Elena A. Morozova; Vsevolod V. Komissarov; Yaroslav V. Tkachev; Vladimir P. Timofeev; Tatyana V. Demidkina; Nicolai G. Faleev (pp. 1247-1256).

A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in ²H₂O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both ¹H- and ¹³C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5′-phosphate (PLP) plane. Consequently, according to Dunathan’s postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with l-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.

Keywords: α-proton exchange; Glycine; Stereospecificity; Methionine γ-lyase; Tyrosine phenol-lyase; Tryptophan indole-lyase; Pyridoxal 5′-phosphate

Sections

Personal tools

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.41, #5)

Sections

Personal tools

Local resources

Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.41, #5)