Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF

YchF, a universally conserved protein, hitherto thought to be a GTPase, was shown to be an ATPase based on structural and biochemical studies on hOLA1, a human ortholog of YchF. However, the cellular role of YchF is unclear. Based on the presence of a RNA binding domain in this protein and significant homology to ribosome binding Obg family GTPases, we examined its ability to associate with the ribosome. Here, we show that Escherichia coli YchF binds the 50S and 70S ribosomal particles in a nucleotide independent manner and it hydrolyzes ATP utilizing a potassium dependent mechanism. A potassium mediated acceleration of hydrolysis activity was thus far known for a few GTPases. Like these, YchF too conserves the structural features required for K⁺ coordination, making it a unique ribosome binding ATPase utilizing a similar mechanism. Furthermore, we show that Lys78 is an important determinant of the potassium dependent ATPase activity.     

Rapid detection of avian influenza H5N1 virus using impedance measurement of immuno-reaction coupled with RBC amplification

Avian influenza virus (AIV) subtype H5N1 was first discovered in the 1990 s and since then its emergence has become a likely source of a global pandemic and economic loss. Currently accepted gold standard methods of influenza detection, viral culture and rRT-PCR, are time consuming, expensive and require special training and laboratory facilities. A rapid, sensitive, and specific screening method is needed for in-field or bedside testing of AI virus to effectively implement quarantines and medications. Therefore, the objective of this study was to improve the specificity and sensitivity of an impedance biosensor that has been developed for the screening of AIV H5. Three major components of the developed biosensor are immunomagnetic nanoparticles for the separation of AI virus, a microfluidic chip for sample control and an interdigitated microelectrode for impedance measurement. In this study polyclonal antibody against N1 subtype was immobilized on the surface of the microelectrode to specifically bind AIV H5N1 to generate more specific impedance signal and chicken red blood cells (RBC) were used as biolabels to attach to AIV H5N1 captured on the microelectrode to amplify impedance signal. RBC amplification was shown to increase the impedance signal change by more than 100% compared to the protocol without RBC biolabels, and was necessary for forming a linear calibration curve for the biosensor. The use of a second antibody against N1 offered much greater specificity and reliability than the previous biosensor protocol. The biosensor was able to detect AIV H5N1 at concentrations down to 10(3) EID(50)ml(-1) in less than 2h.     

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF^FBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF^FBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF^FBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF^FBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development.     

OTX2 Duplication Is Implicated in Hemifacial Microsomia

Hemifacial microsomia (HFM) is the second most common facial anomaly after cleft lip and palate. The phenotype is highly variable and most cases are sporadic. We investigated the disorder in a large pedigree with five affected individuals spanning eight meioses. Whole-exome sequencing results indicated the absence of a pathogenic coding point mutation. A genome-wide survey of segmental variations identified a 1.3 Mb duplication of chromosome 14q22.3 in all affected individuals that was absent in more than 1000 chromosomes of ethnically matched controls. The duplication was absent in seven additional sporadic HFM cases, which is consistent with the known heterogeneity of the disorder. To find the critical gene in the duplicated region, we analyzed signatures of human craniofacial disease networks, mouse expression data, and predictions of dosage sensitivity. All of these approaches implicated OTX2 as the most likely causal gene. Moreover, OTX2 is a known oncogenic driver in medulloblastoma, a condition that was diagnosed in the proband during the course of the study. Our findings suggest a role for OTX2 dosage sensitivity in human craniofacial development and raise the possibility of a shared etiology between a subtype of hemifacial microsomia and medulloblastoma.     

A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col,PLLA/HA,and PLLA/Col for Tissue Engineering Application

A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200–950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.     

Ubiquitylation Pathways In Insulin Signaling and Organismal Homeostasis

The insulin/insulin‐like growth factor‐1 (IGF‐1) signaling (IIS) pathway is a pivotal genetic program regulating cell growth, tissue development, metabolic physiology, and longevity of multicellular organisms. IIS integrates a fine‐tuned cascade of signaling events induced by insulin/IGF‐1, which is precisely controlled by post‐translational modifications. The ubiquitin/proteasome‐system (UPS) influences the functionality of IIS through inducible ubiquitylation pathways that regulate internalization of the insulin/IGF‐1 receptor, the stability of downstream insulin/IGF‐1 signaling targets, and activity of nuclear receptors for control of gene expression. An age‐related decline in UPS activity is often associated with an impairment of IIS, contributing to pathologies such as cancer, diabetes, cardiovascular, and neurodegenerative disorders. Recent findings identified a key role of diverse ubiquitin modifications in insulin signaling decisions, which governs dynamic adaption upon environmental and physiological changes. In this review, we discuss the mutual crosstalk between ubiquitin and insulin signaling pathways in the context of cellular and organismal homeostasis.     

Source and target enzyme signature in serine protease inhibitor active site sequences

Amino acid sequences of proteinaceous proteinase inhibitors have been extensively analysed for deriving information regarding the molecular evolution and functional relationship of these proteins. These sequences have been grouped into several well defined families. It was found that the phylogeny constructed with the sequences corresponding to the exposed loop responsible for inhibition has several branches that resemble those obtained from comparisons using the entire sequence. The major branches of the unrooted tree corresponded to the families to which the inhibitors belonged. Further branching is related to the enzyme specificity of the inhibitor. Examination of the active site loop sequences of trypsin inhibitors revealed that here are strong preferences for specific amino acids at different positions of the loop. These preferences are inhibitor class specific. Inhibitors active against more than one enzyme occur within a class and confirm to class specific sequence in their loops. Hence, only a few positions in the loop seem to determine the specificity. The ability to inhibit the same enzyme by inhibitors that belong to different classes appears to be a result of convergent evolution.     

Toxicity and tolerance of aluminum in plants: tailoring plants to suit to acid soils

Aluminum (Al) stress is one of the serious limiting factors in plant productivity in acidic soils, which constitute about 50 % of the world’s potentially arable lands and causes anywhere between 25 and 80 % of yield losses depending upon the species. The mechanism of Al toxicity and tolerance has been examined in plants, which is vital for crop improvement and enhanced food production in the future. Two mechanisms that facilitate Al tolerance in plants are Al exclusion from the roots and the ability to tolerate Al in the symplast or both. Although efforts have been made to unravel Al-resistant factors, many aspects remain unclear. Certain gene families such as MATE, ALMT, ASR, and ABC transporters have been implicated in some plants for resistance to Al which would enhance the opportunities for creating crop plants suitable to grow in acidic soils. Though QTLs have been identified related to Al-tolerance, no crop plant that is tolerant to Al has been evolved so far using breeding or molecular approaches. The remarkable changes that plants experience at the physiological, biochemical and molecular level under Al stress, the vast array of genes involved in Al toxicity-tolerance, the underlying signaling events and the holistic image of the molecular regulation, and the possibility of creating transgenics for Al tolerance are discussed in this review.