Molecular understanding of sterically controlled compound release through an engineered channel protein (FhuA)

Background  

Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca et al., 2008). Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems.     

Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos

Background  

The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo.     

Predicting changes in protein thermostability brought about by single- or multi-site mutations

Background  

An important aspect of protein design is the ability to predict changes in protein thermostability arising from single- or multi-site mutations. Protein thermostability is reflected in the change in free energy (ΔΔG) of thermal denaturation.     

Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis

Background  

The 19 kDa lipoprotein of Mycobacterium tuberculosis (MTB) is an important target of the innate immune response. To investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19 kDa gene (Δ19) and complemented this strain by reintroduction of the 19 kDa gene into the chromosome as a single copy to produce Δ19::19. We also reintroduced the 19 kDa gene in two modified forms that lacked motifs for acylation (Δ19::19NA) and O-glycosylation (Δ19::19NOG).     

The twoPhysarum polycephalum profiling are not functionally equivalent in yeast cells

Summary Profilin is a ubiquitous actin-monomer-binding protein. The protistPhysarum polycephalum contains two profilins, ProA and ProP, present in amoebae and plasmodia, respectively. We have used mutantSaccharomyces cerevisiae cells in an attempt to observe distinct functions for the two profilins. Profilin-deficient yeast cells (pfy1) have delocalized actin cortical patches, do not contain visible actin cables, have reduced mating efficiency and do not grow at 37 °C or in the presence of caffeine. Deletion of theSRV2 gene (srv2), coding for the adenylyl cyclase-associated protein, also results in an altered actin distribution and an inability to survive on rich medium. We found that the pfy1 and srv2 mutant phenotypes were corrected equally well by the overexpression of Physarum ProA or yeast Pfy1p profilins. The pfy1 cells overexpressing ProP have improved mating efficiency and a normal distribution of actin cortical patches. These cells, however, have barely detectable actin cables, do not grow at 37 °C, and are sensitive to caffeine. Also, the expression of ProP does not correct the growth defect of the srv2 cells. These results suggest that the two Physarum proteins are not functionally equivalent in yeast cells. No difference was detected in the affinity of ProA and ProP for poly-L-proline, while ProA has a slightly greater affinity than ProP for phosphatidylinositol 4,5-biphosphate.Abbreviations FITC tfluorescein isothiocyanate - PIP₂ phosphatidylinositol 4,5-biphosphate - YPD yeast extract peptone dextrose     

Planck-Benzinger Thermal Work Function: Thermodynamic Characterization of the Carboxy-Terminus of p53 Peptide Fragments

The thermodynamic parameters for six p53 carboxy-terminus peptide fragments as determined by analytical ultracentrifugal analysis were compared over the experimental temperature range of 275–310 K to evaluate the Gibbs free energy change as a function of temperature, ΔG ^o (T), from 0 to 400 K using our general linear third-order fitting function, ΔG ^o (T) = α + βT ² + γT ³. Data obtained at the typical experimental temperature range are not sufficient to accurately describe the variations observed in the oligomerization of these p53 fragments. It is necessary to determine a number of thermodynamic parameters, all of which can be precisely assessed using this general third-order linear fitting function. These are the heat of reaction, innate temperature-invariant enthalpy, compensatory temperatures and the thermodynamic molecular switch occurring at the thermal set point. This methodology can be used to distinguish the characteristic structure and stability of p53 carboxy-terminal fragments or other p53 mutants. It should be used for the thermodynamic characterization of any interacting biological system.     

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.     

Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR

Background  

Ranaviruses (family Iridoviridae) are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted protein, designated vIF2α, which shows homology with the eukaryotic translation initiation factor 2α. In analogy to distantly related proteins found in poxviruses vIF2α might act as an inhibitor of the antiviral protein kinase PKR.     

Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide

Background  

The de novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success of these studies relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α, β-dehydroamino acids, especially α, β-dehydrophenylalanine (ΔPhe) comes in use for spawning well-defined structural motifs. Introduction of ΔPhe induces β-bends in small and 3₁₀-helices in longer peptide sequences.     

<Emphasis Type="Italic">Candida albicans SUR7</Emphasis> contributes to secretion,biofilm formation,and macrophage killing

Background  

Candida albicans SUR7 has been shown to be required for plasma membrane organization and cell wall synthesis, but its role in virulence is not known. Using a bioinformatics strategy, we previously identified several novel putative secretion pathway proteins potentially involved in virulence, including the C. albicans homolog of the Saccharomyces cerevisiae endocytosis-related protein Sur7p. We therefore generated a C. albicans sur7Δ null mutant and examined its contribution to key virulence attributes.     

Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant

Background  

EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-ΔN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells.     

Secretion of flagellin by the LEE-encoded type III secretion system of enteropathogenic Escherichia coli

Background  

Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a ΔespADB mutant and a ΔescF T3SS mutant.     

CCR5Δ32 variant and cardiovascular disease in patients with rheumatoid arthritis: a cohort study

Introduction  

The aim of our study was to analyze the influence of the CCR5Δ32 polymorphism in the risk of cardiovascular (CV) events and subclinical atherosclerosis among patients with rheumatoid arthritis (RA).     

Volume-based solvation models out-perform area-based models in combined studies of wild-type and mutated protein-protein interfaces

Background  

Empirical binding models have previously been investigated for the energetics of protein complexation (ΔG models) and for the influence of mutations on complexation (i.e. differences between wild-type and mutant complexes, ΔΔG models). We construct binding models to directly compare these processes, which have generally been studied separately.     

A Δ11 desaturase gene genealogy reveals two divergent allelic classes within the European corn borer (<Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>)

Background  

Moth pheromone mating systems have been characterized at the molecular level, allowing evolutionary biologists to study how changes in protein sequence or gene expression affect pheromone phenotype, patterns of mating, and ultimately, the formation of barriers to gene exchange. Recent studies of Ostrinia pheromones have focused on the diversity of sex pheromone desaturases and their role in the specificity of pheromone production. Here we produce a Δ11 desaturase genealogy within Ostrinia nubilalis. We ask what has been the history of this gene, and whether this history suggests that changes in Δ11 desaturase have been involved in the divergence of the E and Z O. nubilalis pheromone strains.     

Prediction of hot spot residues at protein-protein interfaces by combining machine learning and energy-based methods

Background  

Alanine scanning mutagenesis is a powerful experimental methodology for investigating the structural and energetic characteristics of protein complexes. Individual amino-acids are systematically mutated to alanine and changes in free energy of binding (ΔΔG) measured. Several experiments have shown that protein-protein interactions are critically dependent on just a few residues ("hot spots") at the interface. Hot spots make a dominant contribution to the free energy of binding and if mutated they can disrupt the interaction. As mutagenesis studies require significant experimental efforts, there is a need for accurate and reliable computational methods. Such methods would also add to our understanding of the determinants of affinity and specificity in protein-protein recognition.     

Relationship between endoplasmic reticulum- and Golgi-associated calcium homeostasis and 4-NQO-induced DNA repair in Saccharomyces cerevisiae

Calcium (Ca²⁺) is an important ion that is necessary for the activation of different DNA repair mechanisms. However, the mechanism by which DNA repair and Ca²⁺ homeostasis cooperate remains unclear. We undertook a systems biology approach to verify the relationship between proteins associated with Ca²⁺ homeostasis and DNA repair for Saccharomyces cerevisiae. Our data indicate that Pmr1p, a Ca²⁺ transporter of Golgi complex, interacts with Cod1p, which regulates Ca²⁺ levels in the endoplasmic reticulum (ER), and with Rad4p, which is a nucleotide excision repair (NER) protein. This information was used to construct single and double mutants defective for Pmr1p, Cod1p, and Rad4p followed by cytotoxic, cytostatic, and cell cycle arrest analyses after cell exposure to different concentrations of 4-nitroquinoline 1-oxide (4-NQO). The results indicated that cod1Δ, cod1Δrad4Δ, and cod1Δpmr1Δ strains have an elevated sensitivity to 4-NQO when compared to its wild-type (WT) strain. Moreover, both cod1Δpmr1Δ and cod1Δrad4Δ strains have a strong arrest at G₂/M phases of cell cycle after 4-NQO treatment, while pmr1Δrad4Δ have a similar sensitivity and cell cycle arrest profile when compared to rad4Δ after 4-NQO exposure. Taken together, our results indicate that deletion in Golgi- and ER-associated Ca²⁺ transporters affect the repair of 4-NQO-induced DNA damage.     

The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

Background  

SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).     

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background  

Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.