Systematic characterization of mutations altering protein degradation in human cancers

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Aminopurine based JNK inhibitors for the prevention of ischemia reperfusion injury

In this Letter we describe the optimization of an aminopurine lead (1) with modest potency and poor overall kinase selectivity which led to the identification of a series of potent, selective JNK inhibitors. Improvement in kinase selectivity was enabled by introduction of an aliphatic side chain at the C-2 position. CC-359 (2) was selected as a potential clinical candidate for diseases manifested by ischemia reperfusion injury.     

Fused bicycles as arylketone bioisosteres leading to potent, orally active thiadiazole H3 antagonists

A structure-activity relationship study was undertaken to address the lack of oral exposure of the H3 antagonist 1, which incorporated an arylketone. Among a number of sub-series, the 4H-pyrido[1,2-a]pyrimidin-4-one analog 21 showed an improved PK profile in rat and mouse and was active in an obesity model. The pyrimidin-4-one proved to be a novel and useful ketone bioisostere.     

Cattle MHC nomenclature: is it possible to assign sequences to discrete class I genes?

The cattle major histocompatibility complex (MHC) region contains a variable number of classical class I genes encoding polymorphic molecules involved in antigen presentation. Six classical class I genes have been described, but assigning sequences to these genes has proved problematic. We propose a refinement of the existing nomenclature, which currently names the 97 known classical class I sequences in a single series. Phylogenetic analysis of the 3' portion of the coding region allows segregation of these into six groups; thus, we have prefixed existing names with the appropriate number. Although it is clear that some of these groups correspond to discrete genes, it is currently not possible to state definitively that all do. However, the main groupings are consistent, and in conjunction with other evidence, we feel it is now appropriate to rename the sequences accordingly. Segregation of sequences into groups in this way will facilitate ongoing research and future use of the cattle MHC section of the Immuno Polymorphism Database.     

A transposase strategy for creating libraries of circularly permuted proteins

A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.     

Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata. in vivo effects in rodent brain

An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.     

Modulation of kernel storage proteins in grain sorghum (Sorghum bicolor (L.) Moench)

Sorghum prolamins, termed kafirins, are categorized into subgroups α, β, and γ. The kafirins are co‐translationally translocated to the endoplasmic reticulum (ER) where they are assembled into discrete protein bodies that tend to be poorly digestible with low functionality in food and feed applications. As a means to address the issues surrounding functionality and digestibility in sorghum, we employed a biotechnology approach that is designed to alter protein body structure, with the concomitant synthesis of a co‐protein in the endosperm fraction of the grain. Wherein perturbation of protein body architecture may provide a route to impact digestibility by reducing disulphide bonds about the periphery of the body, while synthesis of a co‐protein, with known functionality attributes, theoretically could impact structure of the protein body through direct association and/or augment end‐use applications of sorghum flour by stabilizing ß‐sheet formation of the kafirins in sorghum dough preparations. This in turn may improve viscoelasticity of sorghum dough. To this end, we report here on the molecular and phenotypic characterizations of transgenic sorghum events that are down‐regulated in γ‐ and the 29‐kDa α‐kafirins and the expression of a wheat Dy10/Dx 5 hybrid high‐molecular weight glutenin protein. The results demonstrate that down‐regulation of γ‐kafirin alone does not alter protein body formation or impacts protein digestibility of cooked flour samples. However, reduction in accumulation of a predicted 29‐kDa α‐kafirin alters the morphology of protein body and enhances protein digestibility in both raw and cooked samples.     

Sequences of a hairpin structure in the 3'-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10(-7) M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3'-untranslated region (3'-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilization of mRNA. Sequential substitution mutagenesis of the 7.6S region indicates that a 90-nt region is required for DEX-mediated stabilization and maintenance of intrinsic stability. In this region, one 30-nt-long element (002), predicted to form a stem-loop structure, is sufficient for DEX-mediated stabilization of mRNA and intrinsic mRNA stability. Cytosolic proteins specifically bind element 002, and binding activity is unaffected whether proteins are isolated from cells incubated in the absence or presence of DEX. While loop sequences of element 002 have no role in regulation of SP-B mRNA stability, the proximal stem sequences are required for DEX-mediated stabilization and specific binding of proteins. Mutation of the sequences that comprise the proximal or distal arm of the stem negates the destabilizing activity of element 002 on intrinsic SP-B mRNA stability. These results indicate that cytosolic proteins bind a single hairpin structure that mediates intrinsic and hormonal regulation of SP-B mRNA stability via mechanisms that involve sequences of the stems of the hairpin structure.     

Optimizing the refolding conditions of self-assembling polypeptide nanoparticles that serve as repetitive antigen display systems

Nanoparticles show great promise as potent vaccine candidates since they are readily taken up by the antigen presenting cells of the immune system. The particle size and the density of the B cell epitopes on the surface of the particles greatly influences the strength of the humoral immune response. We have developed a novel type of nanoparticle composed of peptide building blocks (Raman et al., 2006) and have used such particles to design vaccines against malaria and SARS (Kaba et al., 2009; Pimentel et al., 2009). Here we investigate the biophysical properties and the refolding conditions of a prototype of these self-assembling polypeptide nanoparticles (SAPNs). SAPNs are formed from a peptide containing a pentameric and a trimeric coiled-coil domain. At near physiological conditions the peptide self-assembles into about 27 nm, roughly spherical SAPNs. The average size of the SAPNs increases with the salt concentration. The optimal pH for their formation is between 7.5 and 8.5, while aggregation occurs at lower and higher values. A glycerol concentration of about 5% v/v is required for the formation of SAPNs with regular spherical shapes. These studies will help to optimize the immunological properties of SAPNs.     

Cytokine Interactions with Adrenal Medullary Chromaffin Cells

It is generally accepted that a bi-directional or reciprocal interaction occurs between the immune and neuroendocrine systems, and that this relationship is important for the appropriate physiological functioning of both systems. Similarly, an imbalance in this relationship may contribute to a number of pathologies, most notably those relating to stress. The aim of this article is to consider the interaction of cytokines with the adrenal medulla, a potentially important player in this relationship. The chromaffin cells of the adrenal medulla release catecholamines and a range of biologically active peptides in response to a wide variety of stress-related signals. A growing body of evidence indicates that this stress response is influenced by, and in turn has influence upon, immune signalling. This brief review will focus primarily on the best-described adrenal medullary active cytokines, namely interferon-α, interleukin-6, interleukin-1α/β and tumour necrosis factor-α. In each case, three key issues will be addressed: the physiologically relevant source of the cytokine; the intracellular signalling events arising from activation of its receptor and finally the cellular consequences of such activation in terms of modulation of gene expression and the secretory output of the chromaffin cells.