Stem-cell-abundant proteins Nanog, Nucleostemin and Musashi1 are highly expressed in malignant cervical epithelial cells

<p>Abstract</p> <p>Background</p> <p>Nanog, nucleostemin (NS) and musashi1 (Msi1) are proteins that are highly expressed in undifferentiated embryonic stem (ES) cells and have been shown to be essential in maintaining the pluripotency and regulating the proliferation and asymmetric division of ES cells and several nervous system tumor cells. The roles of Nanog, NS and Msi1 in development and progression of cervical carcinoma have, until now, not been well documented.</p> <p>Methods</p> <p>In this study, expression of Nanog, NS and Msi1 was detected by immunohistochemistry analysis in 235 patients with various degrees of cervical epithelial lesions, including 49 with normal cervical epithelia, 31 with mild dysplasia (CIN I), 77 with moderate-severe dysplasia (CIN II-III) and 78 with squamous cervical carcinomas (SCCs). Associations with various clinical pathological prognostic variables were analyzed in 50 early-stage SCC patients.</p> <p>Results</p> <p>Nanog, NS and Msi1 expression levels were significantly higher in SCC patients compared with CIN patients, and were higher in CIN patients compared with those with normal cervical epithelia. Nanog expression levels showed significantly differences according to different tumor sizes (P < 0.05), whereas there were no differences in NS and Msi1 expression levels according to different clinical pathological parameters.</p> <p>Conclusion</p> <p>Our findings indicate that Nanog, NS and Msi1 may be involved in carcinogenesis of the cervix and progression of cervical carcinoma.</p

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell ‘stemness’ and tumorigenesis. Msi1 reportedly binds to the 3′-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between β1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins

Methyl group assignment using pseudocontact shifts with PARAssign

Macromolecular Biochemistr

Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method

Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated using NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of staphylococcal nuclease (SNase), which contains 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of ∼ 5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereospecifically assigned using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys. The complete set of assigned 1H, 13C, and 15N NMR signals for the Lys side-chain moieties affords useful structural information. For example, the set includes the characteristic chemical shifts for the 13Cδ, 13Cε, and 15Nζ signals for Lys-66, which has the deprotonated ζ-amino group, and the large upfield shifts for the 1H and 13C signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are indicative of nearby aromatic rings. The 13Cε and 15Nζ chemical shifts of the SNase variant selectively labeled with either [ε-13C;ε,ε-D2]-Lys or SAIL-Lys, dissolved in H2O and D2O, showed that the deuterium-induced shifts for Lys-66 were substantially different from those of the other 20 Lys residues. Namely, the deuterium-induced shifts of the 13Cε and 15Nζ signals depend on the ionization states of the ζ-amino group, i.e., −0.32 ppm for Δδ13Cε [NζD3+-NζH3+] vs. −0.21 ppm for Δδ13Cε [NζD2-NζH2] and −1.1 ppm for Δδ15Nζ[NζD3+-NζH3+] vs. −1.8 ppm for Δδ15Nζ[NζD2-NζH2]. Since the 1D 13C NMR spectrum of a protein selectively labeled with [ε-13C;ε,ε-D2]-Lys shows narrow (&gt; 2 Hz) and well-dispersed 13C signals, the deuterium-induced shift difference of 0.11 ppm for the protonated and deprotonated ζ-amino groups, which corresponds to 16.5 Hz at a field strength of 14 T (150 MHz for 13C), could be accurately measured. Although the isotope shift difference itself may not be absolutely decisive to distinguish the ionization state of the ζ-amino group, the 13Cδ, 13Cε, and 15Nζ signals for a Lys residue with a deprotonated ζ-amino group are likely to exhibit distinctive chemical shifts as compared to the normal residues with protonated ζ-amino groups. Therefore, the isotope shifts would provide a useful auxiliary index for identifying Lys residues with deprotonated ζ-amino groups at physiological pH levels.</p

Highly Dispersed 3C Silicon Carbide Nanoparticles with a Polydopamine/Polyglycerol Shell for Versatile Functionalization

Silicon carbide (SiC) nanoparticles containing lattice defects are attracting considerable attention as next-generation imaging probes and quantum sensors for visualizing and sensing life activities. However, SiC nanoparticles are not currently used in biomedical applications because of the lack of technology for controlling their physicochemical properties. Therefore, in this study, SiC nanoparticles are deaggregated, surface-coated, functionalized, and selectively labeled to biomolecules of interest. A thermal-oxidation chemical-etching method is developed for deaggregating and producing a high yield of dispersed metal-contaminant-free SiC nanoparticles. We further demonstrated a polydopamine coating with controllable thickness that can be used as a platform for decorating gold nanoparticles on the surface, enabling photothermal application. We also demonstrated a polyglycerol coating, which gives excellent dispersity to SiC nanoparticles. Furthermore, a single-pot method is developed to produce mono/multifunctional polyglycerol-modified SiC nanoparticles. Using this method, CD44 proteins on cell surfaces are selectively labeled through biotin-mediated immunostaining. The methods developed in this study are fundamental for applying SiC nanoparticles to biomedical applications and should considerably accelerate the development of various SiC nanoparticles to exploit their potential applications in bioimaging and biosensing

Myosin VI Undergoes Cargo-Mediated Dimerization

Myosin VI is the only known molecular motor that moves toward the minus ends of actin filaments; thus, it plays unique roles in diverse cellular processes. The processive walking of myosin VI on actin filaments requires dimerization of the motor, but the protein can also function as a nonprocessive monomer. The molecular mechanism governing the monomer-dimer conversion is not clear. We report the high-resolution NMR structure of the cargo-free myosin VI cargo-binding domain (CBD) and show that it is a stable monomer in solution. The myosin VI CBD binds to a fragment of the clathrin-coated vesicle adaptor Dab2 with a high affinity, and the X-ray structure of the myosin VI CBD in complex with Dab2 reveals that the motor undergoes a cargo-binding-mediated dimerization. The cargo-binding-induced dimerization may represent a general paradigm for the regulation of processivity for myosin VI as well as other myosins, including myosin VII and myosin X

Soluble domains of cytochrome c-556 and Rieske iron–sulfur protein from Chlorobaculum tepidum: Crystal structures and interaction analysis

In photosynthetic green sulfur bacteria, the electron transfer reaction from menaquinol:cytochrome c oxidoreductase to the P840 reaction center (RC) complex occurs directly without any involvement of soluble electron carrier protein(s). X-ray crystallography has determined the three-dimensional structures of the soluble domains of the CT0073 gene product and Rieske iron-sulfur protein (ISP). The former is a mono-heme cytochrome c with an α-absorption peak at 556 nm. The overall fold of the soluble domain of cytochrome c-556 (designated as cyt c-556sol) consists of four α-helices and is very similar to that of water-soluble cyt c-554 that independently functions as an electron donor to the P840 RC complex. However, the latter's remarkably long and flexible loop between the α3 and α4 helices seems to make it impossible to be a substitute for the former. The structure of the soluble domain of the Rieske ISP (Rieskesol protein) shows a typical β-sheets-dominated fold with a small cluster-binding and a large subdomain. The architecture of the Rieskesol protein is bilobal and belongs to those of b6f-type Rieske ISPs. Nuclear magnetic resonance (NMR) measurements revealed weak non-polar but specific interaction sites on Rieskesol protein when mixed with cyt c-556sol. Therefore, menaquinol:cytochrome c oxidoreductase in green sulfur bacteria features a Rieske/cytb complex tightly associated with membrane-anchored cyt c-556