Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.

Inhibition of Mycobacterium tuberculosis RNA Polymerase by Binding of a Gre Factor Homo log to the Secondary Channel

Because of its essential nature, each step of transcription, viz., initiation, elongation, and termination, is subjected to elaborate regulation. A number of transcription factors modulate the rates of transcription at these different steps, and several inhibitors shut down the process. Many modulators, including small molecules and proteinaceous inhibitors, bind the RNA polymerase (RNAP) secondary channel to control transcription. We describe here the first small protein inhibitor of transcription in Mycobacterium tuberculosis. Rv3788 is a homolog of the Gre factors that binds near the secondary channel of RNAP to inhibit transcription. The factor also affected the action of guanosine pentaphosphate (pppGpp) on transcription and abrogated Gre action, indicating its function in the modulation of the catalytic center of RNAP. Although it has a Gre factor-like domain organization with the conserved acidic residues in the N terminus and retains interaction with RNAP, the factor did not show any transcript cleavage stimulatory activity. Unlike Rv3788, another Gre homolog from Mycobacterium smegmatis, MSMEG_6292 did not exhibit transcription-inhibitory activities, hinting at the importance of the former in influencing the lifestyle of M. tuberculosis.

Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16

We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsinf-old and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. (C) 2013 The Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.

Guanylyl Cyclase C Receptor: Regulation of Catalytic Activity by ATP

Guanylyl cyclase C (GCC), a member of the family of membrane bound guanylyl cyclases is the receptor for the heat-stable enterotoxin (ST) peptides and the guanylin family of endogenous peptides. GCC is activated upon ligand binding to increase intracellular cGMP levels, which in turn activates other downstream signalling events in the cell. GCC is also activated in vitro by nonionic detergents. We have used the T84 cell line as a model system to investigate the regulation of GCC activity by ATP. Ligand-stimulated GCC activity is potentiated in the presence of ATP, whereas detergent-stimulated activity is inhibited. The potentiation of GCC activity by ATP is dependent on the presence of Mg2+ ions, and is probably brought about by a direct binding of Mg-ATP to GCC. The protein kinase-like domain of GCC, which has earlier been shown to play a critical role in the regulation of GCC activity, may be a possible site for the binding of Mg-ATP to GCC.

Unstructured N Terminus of the RNA Polymerase II Subunit Rpb4 Contributes to the Interaction of Rpb4·Rpb7 Subcomplex with the Core RNA Polymerase II of Saccharomyces cerevisiae

Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4 center dot Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.

Functional Significance of an Evolutionarily Conserved Alanine (GCA) Resume Codon in tmRNA in Escherichia coli

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.

Magmas functions as a ROS regulator and provides cytoprotection against oxidative stress-mediated damages

Redox imbalance generates multiple cellular damages leading to oxidative stress-mediated pathological conditions such as neurodegenerative diseases and cancer progression. Therefore, maintenance of reactive oxygen species (ROS) homeostasis is most important that involves well-defined antioxidant machinery. In the present study, we have identified for the first time a component of mammalian protein translocation machinery Magmas to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues and cancer cells that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance toward oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of electron transport chain (ETC) complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as a ROS sensor was found to be independent of its role in protein import. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cell tolerance to oxidative stress even in yeast model organism. The cytoprotective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress-related diseases.

A Smooth Discretization Bridging Finite Element and Mesh-free Methods Using Polynomial Reproducing Simplex Splines

This work sets forth a `hybrid' discretization scheme utilizing bivariate simplex splines as kernels in a polynomial reproducing scheme constructed over a conventional Finite Element Method (FEM)-like domain discretization based on Delaunay triangulation. Careful construction of the simplex spline knotset ensures the success of the polynomial reproduction procedure at all points in the domain of interest, a significant advancement over its precursor, the DMS-FEM. The shape functions in the proposed method inherit the global continuity (Cp-1) and local supports of the simplex splines of degree p. In the proposed scheme, the triangles comprising the domain discretization also serve as background cells for numerical integration which here are near-aligned to the supports of the shape functions (and their intersections), thus considerably ameliorating an oft-cited source of inaccuracy in the numerical integration of mesh-free (MF) schemes. Numerical experiments show the proposed method requires lower order quadrature rules for accurate evaluation of integrals in the Galerkin weak form. Numerical demonstrations of optimal convergence rates for a few test cases are given and the method is also implemented to compute crack-tip fields in a gradient-enhanced elasticity model.

Spontaneous and reversible self-assembly of a polypeptide fragment of insulin-like growth factor binding protein-2 into fluorescent nanotubular structures

In this communication, we report the spontaneous and reversible in vitro self-assembly of a polypeptide fragment derived from the C-terminal domain of Insulin-like Growth Factor Binding Protein (IGFBP-2) into soluble nanotubular structures several micrometres long via a mechanism involving inter-molecular disulfide bonds and exhibiting enhanced fluorescence.

A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

Constraining the low-energy pion electromagnetic form factor with space-like and phase of time-like data

The Taylor coefficients c and d of the EM form factor of the pion are constrained using analyticity, knowledge of the phase of the form factor in the time-like region, 4m(pi)(2) <= t <= t(in) and its value at one space-like point, using as input the (g - 2) of the muon. This is achieved using the technique of Lagrange multipliers, which gives a transparent expression for the corresponding bounds. We present a detailed study of the sensitivity of the bounds to the choice of time-like phase and errors present in the space-like data, taken from recent experiments. We find that our results constrain c stringently. We compare our results with those in the literature and find agreement with the chiral perturbation-theory results for c. We obtain d similar to O(10) GeV-6 when c is set to the chiral perturbation-theory values.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Defining the pathway to insulin-like growth factor system targeting in cancer

The insulin-like growth factors (IGEs; IGF-1 and IGF-2) play central roles in cell growth, differentiation, survival, transformation and metastasis. The biologic effects of the IGFs are mediated by the IGF-1 receptor (IGF-1R), a receptor tyrosine kinase with homology to the insulin receptor (IR). Dysregulation of the ICE system is well recognized as a key contributor to the progression of multiple cancers, with IGF-1R activation increasing the tumorigenic potential of breast, prostate, lung, colon and head and neck squamous cell carcinoma (HNSCC). Despite this relationship, targeting the IGF-1R has only recently undergone development as a molecular cancer therapeutic. As it has taken hold, we are witnessing a robust increase and interest in targeting the inhibition of IGF-1R signaling. This is accentuated by the list of over 30 drugs, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) that are under evaluation as single agents or in combination therapies 1]. The ICE-binding proteins (IGFBPs) represent the third component of the ICE system consisting of a class of six soluble secretory proteins. They represent a unique class of naturally occurring ICE-antagonists that bind to and sequester IGF-1 and IGF-2, inhibiting their access to the IGF-1R. Due to their dual targeting of the IGFs without affecting insulin action, the IGFBPs are an untapped ``third'' class of IGF-1R inhibitors. in this commentary, we highlight some of the significant aspects of and prospects for targeting the IGF-1R and describe what the future may hold. (C) 2010 Elsevier Inc. All rights reserved.

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160–455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the [beta]-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between [beta] strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

Cooperative Regulation of NOTCH1 Protein-Phosphatidylinositol 3-Kinase (PI3K) Signaling by NOD1, NOD2, and TLR2 Receptors Renders Enhanced Refractoriness to Transforming Growth Factor-beta (TGF-beta)- or Cytotoxic T-lymphocyte Antigen 4 (CTLA-4)-mediated Impairment of Human Dendritic Cell Maturation

Dendritic cells (DCs) as sentinels of the immune system are important for eliciting both primary and secondary immune responses to a plethora of microbial pathogens. Cooperative stimulation of a complex set of pattern-recognition receptors, including TLR2 and nucleotide-binding oligomerization domain (NOD)-like receptors on DCs, acts as a rate-limiting factor in determining the initiation and mounting of the robust immune response. It underscores the need for ``decoding'' these multiple receptor interactions. In this study, we demonstrate that TLR2 and NOD receptors cooperatively regulate functional maturation of human DCs. Intriguingly, synergistic stimulation of TLR2 and NOD receptors renders enhanced refractoriness to TGF-beta- or CTLA-4-mediated impairment of human DC maturation. Signaling perturbation data suggest that NOTCH1-PI3K signaling dynamics assume critical importance in TLR2- and NOD receptor-mediated surmounting of CTLA-4- and TGF-beta -suppressed maturation of human DCs. Interestingly, the NOTCH1-PI3K signaling axis holds the capacity to regulate DC functions by virtue of PKC delta-MAPK-dependent activation of NF-kappa B. This study provides mechanistic and functional insights into TLR2-and NOD receptor-mediated regulation of DC functions and unravels NOTCH1-PI3K as a signaling cohort for TLR2 and NOD receptors. These findings serve in building a conceptual foundation for the design of improved strategies for adjuvants and immunotherapies against infectious diseases.