Exploring protein-protein interactions at the proteome level

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Predicting zinc binding at the proteome level

Background  

Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biological function, for regulation of their activities or for structural purposes. Metal-binding properties remain difficult to predict as well as to investigate experimentally at the whole-proteome level. Consequently, the current knowledge about metalloproteins is only partial.     

Order from chaos: observing hormesis at the proteome level

We report on an observed regularity in the overall response of cell proteome under influence of different doses of a peroxisome proliferator. Our analysis for the first time demonstrates presence of hormesis at the cellular level. It is shown that despite the fact that the response of individual proteins remains unpredictable, the perturbation of proteome as a whole (measured as the average departure of protein abundances from the corresponding values of the control group) shows regularity: It initially decreases, reaches a minimum, and then increases as the concentration of the used proliferator continue to increase. The work is based on the available data of Anderson at al. on the effects of peroxisome proliferator LY171883 on protein abundances in mouse liver when administrated at different concentrations.     

Insights into Monascus biology at the genetic level

The genus of Monascus was nominated by van Tieghem in 1884, but its fermented product—red mold rice (RMR), namely red yeast rice, has been used as folk medicines, food colorants, and fermentation starters for more than thousands of years in oriental countries. Nowadays, RMR is widely developed as food supplements around the world due to its functional compounds such as monacolin K (MK, also called lovastatin) and γ-aminobutyric acid. But the usage of RMR also incurs controversy resulting from contamination of citrinin (a kind of mycotoxin) produced by some Monascus strains. In the past decade, it has made great progress to Monascus spp. at the genetic level with the application of molecular biology techniques to restrain the citrinin production and increase the yields of MK and pigment in RMR, as well as aid Monascus classification and phylogenesis. Up to now, hundreds of papers about Monascus molecular biology (MMB) have been published in the international primary journals. However, to our knowledge, there is no MMB review issued until now. In this review, current understanding of Monascus spp. from the view of molecular biology will be covered and insights into research areas that need to be further investigated will also be discussed.     

Investigating the biology of consciousness.

The fact that consciousness is a private, first-person phenomenon makes it more difficult to study than other cognitive phenomena that, although being equally private, also have characteristic behavioural signatures. Nonetheless, by combining cognitive and neurobiological methods, it is possible to approach consciousness, to describe its cognitive nature, its behavioural correlates, its possible evolutionary origin and functional role; last but not least, it is possible to investigate its neuroanatomical and neurophysiological underpinnings. In this brief essay I distinguish between two kinds of consciousness: core consciousness and extended consciousness. Core consciousness corresponds to the transient process that is incessantly generated relative to any object with which an organism interacts, and during which a transient core self and transient sense of knowing are automatically generated. Core consciousness requires neither language nor working memory, and needs only a brief short-term memory. Extended consciousness is a more complex process. It depends on the gradual build-up of an autobiographical self, a set of conceptual memories pertaining to both past and anticipated experiences of an individual, and it requires conventional memory. Extended consciousness is enhanced by language.     

The biology of breast cancer at the cellular level

Two properties seem fundamental to cancer; heterogeneity and progression (Foulds (1975) Academic Press, New York; Heppner et al. (1979) Commentaries on Research in Breast Disease, Vol. 1 (Bulbrook, R. and Taylor, D.J., eds.), pp. 177-191, Plenum Press, New York). Relatively little is understood about the premalignant stages of human breast disease in vivo. When the disease manifests as invasive carcinoma, its behavior exhibits great diversity, sometimes metastasizing rapidly, while in other cases 10-30 years pass before metastases proliferate. Here we review various aspects of breast cancer in vivo and consider how they predict properties of breast cancer found in culture. All of the experiments are consistent with the hypothesis proposed by Nowell (1976) Science 194, 23-28, that a fundamental aspect of malignancy is an increased genetic instability and that many of the cells within tumors are nonviable results of genetic instability. We suggest that most of the viable cells within primary breast carcinomas are diploid and are not yet capable of aspects of metastatic spread. What these cells have attained is an increased propensity for genetic instability which enables them to generate randomly aneuploid but frequently lethal genetic configurations. Occasionally one of these altered genomes is associated with the ability to proliferate at a metastatic site. This hypothesis implies that metastases from various patients may have arisen by divergent pathways and may also be divergent in many other aspects of their physiology, unrelated to malignancy. Such extreme heterogeneity may hamper attempts to understand fundamental aspects of malignancy. Hence we suggest that the less anaplastic and less divergent diploid cells within the primary carcinomas might be an important resource to gain insights into the critical alterations that are responsible for initiating frankly malignant behavior.     

Structural analysis of mammalian protein phosphorylation at a proteome level

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Response of Pseudomonas putida KT2440 to phenol at the level of membrane proteome

This study led to the extension and refinement of our current model for the global response of Pseudomonas putida KT2440 to phenol by getting insights into the adaptive response mechanisms involving the membrane proteome. A two-dimensional gel electrophoresis based protocol was optimized to allow the quantitative comparison of membrane proteins, by combining inner and outer membrane fractionation with membrane protein solubilization using the detergent dodecylmaltoside. Following phenol exposure, a coordinate increased content of protein subunits of known or putative solvent efflux pump systems (e.g. TtgA, TtgC, Ttg2A, Ttg2C, and PP_1516-7) and a decreased content of porins OprB, OprF, OprG and OprQ was registered, consistent with an adaptive response to reduce phenol intracellular concentration. This adaptive response may in part be mediated by post-translational modifications, as suggested by the relative content of the multiple forms identified for a few porins and efflux pump subunits. Results also suggest the important role of protein chaperones, of cell envelope and cell surface and of a more active respiratory chain in the response to phenol. All these mechanistic insights may be extended to Pseudomonas adaptation to solvents, of possible impact in biodegradation, bioremediation and biocatalysis.     

Portrait of a pathogen: the Mycobacterium tuberculosis proteome in vivo

Background

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo.

Methodology/Principal Findings

We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection.

Conclusions/Significance

Cumulatively, the results of this work may provide the basis for rational drug design – identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.     

Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry

The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.     

Project work in social biology at GCE Advanced level

The Cambridge Advanced level GCE Examination¹ in Social Biology includes an individual study (project). The system by which project topics are submitted, modified, and approved is outlined in this paper and an indication is given of the standard and quantity of work expected. The criteria on which assessment is based are explained, the range of individual studies is summarized, and the cases for and against project work are given.     

High-throughput sequencing for the study of bacterial pathogen biology

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Complementary analysis of the vegetative membrane proteome of the human pathogen Staphylococcus aureus

The Gram-positive bacterium Staphylococcus aureus is a serious human pathogen causing a wide variety of diseases, and its increasing resistance toward all available antibiotics makes its further investigation absolutely essential. We examined the membrane proteome of exponentially growing cells of S. aureus COL because this subproteome plays a major role in the virulence of the bacterium in its host. In general, an analysis of membrane proteins is impeded by their hydrophobic nature as well as by a high abundance of many cytosolic proteins. The implementation of three different technologies, one-dimensional gel-LC, two-dimensional LC, and a membrane shaving approach combined with MS/MS analyses, enabled an identification of 271 integral and 86 peripheral membrane proteins from exponentially growing cells. In particular, the latter approach that combined membrane shaving with a subsequent chymotrypsin digest of integral membrane domains of proteins greatly facilitated the detection of hydrophobic peptides derived from membrane-spanning segments (713 peptides, 60% of all peptides) and therefore yielded almost exclusively highly hydrophobic integral membrane proteins (96.7%). A comparison of the various methods disclosed the one-dimensional gel-LC and the shaving approach to be highly complementary techniques. A combination of them will reveal a most comprehensive view on membrane proteomes.     

Saccharomyces cerevisiae in the stationary phase as a model organism--characterization at cellular and proteome level

The yeast Saccharomyces cerevisiae has been used as a model organism to investigate responses to different environmental stressors. The importance of their conclusions has been expanded to human cells. The experiments were done with exponentially growing cells, which do not resemble human cells. Human and other eukaryotic cells spend the greater part of their lives in a quiescent state, known as G0 corresponding to the yeast stationary phase. Providing energy, which comes from mitochondrial respiration, is also common. Thus, in the present study S. cerevisiae was used in the stationary phase for characterization at the cellular and proteome levels. At the cellular level, optical density, cell viability, glycogen content, intracellular oxidation and cell energy metabolic activity were measured, while at the proteome level, protein profiles were analyzed using two-dimensional electrophoresis. The data obtained at both levels provide better insight into quiescence program state, which still remains poorly understood. At their base, optimal time period reflecting a stable metabolic and oxidative state of the yeast was determined. Consequently, this period is the appropriate to study changes in cell oxidant status and energy metabolic activity in response to different environmental stressors.