Single Protein Level Research Articles

A Large Number of Protein Expression Changes Occur Early in Life and Precede Phenotype Onset in a Mouse Model for Huntington Disease

Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.

From genotype to phenotype: a longitudinal study of a patient with hypertrophic cardiomyopathy due to a mutation in the MYBPC3 gene

Many of the links between the genotype and phenotype in hypertrophic cardiomyopathy remain unexplained. In this unique longitudinal study we have investigated a patient with classical clinical phenotypic features of hypertrophic obstructive cardiomyopathy, with a known mutation in MYBPC3, the most commonly affected gene in this disease. By collecting cardiac tissue from the patient at the time of surgical myectomy for relief of left ventricular outflow tract obstruction, we have been able to examine the structure of the myocytes and the functional differences that occur in MyBP-C mutated HCM cardiac tissue from single protein level, onto single cardiomyocyte contractility, through to whole organ function as assessed clinically by echocardiography.

Matrix metalloproteinases and their tissue inhibitors as prognostic indicators for diagnostic and surgical recurrence in Crohnʼs disease

Recurrence of disease after surgically induced remission constitutes a major and largely unpredictable problem in Crohn's disease (CD). Matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMP) are involved in the (etio)pathogenesis of CD and may thereby also affect postsurgical outcome. We studied the predictive value of 1) allelic composition at MMP, TIMP, and TNF-alpha single nucleotide polymorphism loci, and 2) MMP and TIMP intestinal protein levels relative to important clinical variables for recurrence of CD after resection of diseased bowel. From 87 CD patients with a full medical record, surgically resected tissue was homogenized and analyzed for single nucleotide polymorphism (SNP) genotype and MMP-TIMP protein levels. The prognostic value of these parameters was determined using the uni- and multivariate Cox proportional hazards analyses. The T allele at TIMP-1 SNP +372 T/C was found to be associated with an increased risk for surgical recurrence. Higher levels of TIMP-1, TIMP-2, and MMP-9 in noninflamed CD tissue, but not in inflamed tissue, and negative smoking status independently protected against diagnostic and/or surgical recurrence. The TIMP-1 SNP +372 T allele with an increased risk of recurrence is in line with our previous results demonstrating increased CD susceptibility and low TIMP-1 protein expression associated with this allele. High TIMP and MMP-9 levels in noninflamed tissue are predictive of a favorable disease recurrence in CD. The contribution of MMP-9 and TIMPs to disease recurrence appears not to be mediated by smoking status, since no correlation with this parameter could be demonstrated.

Human Cataract Lens Membrane at Subnanometer Resolution

Human pathologies often originate from molecular disorders. Therefore, imaging technology as one of the bases for the identification and understanding of pathologies must provide views of single molecules at subnanometer resolution. Membrane proteins mediate many of life's most important processes, and their malfunction is often lethal or leads to severe disease. The membrane proteins aquaporin-0 (AQP0) and connexons form junctional microdomains between healthy lens core cells in which AQP0 form square arrays surrounded by connexons. Malfunction of both proteins results in the formation of cataract. We have used high-resolution atomic force microscopy (AFM) to image junctional microdomains in membranes from an individual human eye lens with senile cataract. Images at subnanometer resolution report individual helix-connecting loops of four amino acid residues on the AQP0 surface. We describe the supramolecular assembly and the conformational state of AQP0 in junctional microdomains, where a mixture of truncated junctional and full-length water channel AQP0 form square arrays. Imaging of microdomain borders revealed individual AQP0 tetramers and no associated connexon, indicating a lack of metabolite transport, waste accumulation, and enlarged regions of non-adhering membranes, causing cataract in this individual. This first high-resolution view of the membrane of this pathological human tissue provides insights into cataract pathology at the single membrane protein level, and indicates the power of the AFM as a future tool in medical imaging at subnanometer resolution.

Direct Nanopatterning of 3D Chemically Active Structures for Biological Applications

Methods that allow the production of patterned surfaces at the nanometer scale are powerful tools for studying fundamental aspects of cell adhesion at the level of single protein/ receptor molecules, [1] tissue engineering, [2] and cellular biosensing. [3] Furthermore, patterning of biomolecules is also a challenging issue for the implementation of a new generation of miniaturized biochips requiring smaller reagent quantities with lower detection limits. Nanopatterned surfaces have already demonstrated the potential to fulfill these requirements, and recent results have indicated that a strong increase in the detection sensitivity of immunosensors was obtained when antibodies were immobilized on nanostructured surfaces. [4]

The 'evolvability' of promiscuous protein functions

How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level? Results from directed laboratory evolution experiments indicate that the evolution of a new function is driven by mutations that have little effect on the native function but large effects on the promiscuous functions that serve as starting point. Thus, an evolving protein can initially acquire increased fitness for a new function without losing its original function. Gene duplication and the divergence of a completely new protein may then follow.

A quantitative model for presynaptic free Ca2+ dynamics during different stimulation protocols

The presynaptic free Ca2+ dynamics of neurons due to various stimulation protocols is investigated in a mathematical model. The model includes Ca2+ influx through voltage-dependent Ca2+ channels, Ca2+ buffering by endogenous and exogenous buffers as well as Ca2+ efflux through ATP-driven plasma membrane Ca2+ pumps and Na+/Ca2+ exchangers. We want to support a specific way of modeling which starts on the level of single proteins. Each protein is represented by characteristics that are determined by single protein experiments and that are considered to be widely independent of neuron types. This allows the applications of the model to different classes of neurons and experiments. The procedure is demonstrated for single boutons of pyramidal neurons of the rat neocortex. The corresponding fluorescence measurements of Koester and Sakmann (J. Physiol. 529 (2000) 625) are quantitatively reproduced. The model enables us to reconstruct the free Ca2+ dynamics in neurons as it would have been without fluorescence indicators starting from the fluorescence data. We discuss the different Ca2+ responses and find that during high-frequency stimulation an accumulation of free Ca2+ occurs above some threshold stimulation frequency. The threshold frequency depends on the amount of fluorescence indicator used in the experiment.

Analysing the action of bacterial toxins in living cells with fluorescence resonance energy transfer (FRET)

AbstractBacterial toxins represent small molecules produced by microorganisms. Different toxins act on specific target molecules in mammalian cells. Once discovered, bacterial toxins have been providing tools to study cellular functions and often helped the dissection of complex cellular pathways, e.g. endocytic or secretory trafficking or signal transduction, by virtue of the fact that they either block or activate their specific cellular target molecules. Purified bacterial toxins have also allowed to address many basic biological questions and have provided tools for in vitro and in vivo experimental approaches in many fields of modern biology. The understanding of how bacterial toxins act in living cells often depends on our ability to visualize the trafficking and signaling pathways of these molecules. Fluorescence microscopy and other imaging tools are essential to provide insights into the functional changes induced by these pathogens at the level of individual host cells or single target proteins. Inside a single cell we can measure and quantify the effects of bacterial toxins on specific cellular proteins by microscopic and spectroscopic techniques. Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows to study protein-protein interactions. FRET can provide distance information in the range of 3 – 7 nm between fluorescently labeled bacterial proteins in the live cell and cellular target proteins expressed as chimeras with green fluorescent protein (GFP), or spectrally shifted variants thereof. The purpose of this review is to introduce readers to the main experimental setups for analyses of protein-protein interactions using FRET as well as some applications.

Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.

Is a single time point C-reactive protein predictive of outcome in peritoneal dialysis patients?

C-reactive protein is the prototype marker of inflammation and has been shown to predict mortality in hemodialysis patients. However, it remains uncertain as to whether a single C-reactive protein level has similar prognostic significance in peritoneal dialysis patients. A single high-sensitivity C-reactive protein (hs-CRP) level was measured in 246 continuous ambulatory peritoneal dialysis patients without active infections at study baseline together with indices of dialysis adequacy, echocardiographic parameters (left ventricular mass index, left ventricular dimensions, and ejection fraction), nutrition markers (serum albumin, dietary intake, and subjective global assessment) and biochemical parameters (hemoglobin, lipids, calcium, and phosphate). The cohort was then followed-up prospectively for a median of 24 mo (range, 2 to 34 mo), and outcomes were studied in relation to these parameters. Fifty-nine patients died (36 from cardiovascular causes) during the follow-up period. The median hs-CRP level was 2.84 mg/L (range, 0.20 to 94.24 mg/L). Patients were stratified into tertiles according to baseline hs-CRP, namely those with hs-CRP < or = 1.26 mg/L, 1.27 to 5.54 mg/L, and > or = 5.55 mg/L. Those with higher hs-CRP were significantly older (P < 0.001), had greater body mass index (P < 0.001), higher prevalence of coronary artery disease (P = 0.003), and greater left ventricular mass index (P < 0.001). One-year overall mortality was 3.9% (lower) versus 8.8% (middle) versus 21.3% (upper tertile) (P < 0.0001). Cardiovascular death rate was 2.7% (lower) versus 5.2% (middle) versus 16.2% (upper tertile) (P < 0.0001). Multivariable Cox regression analysis showed that every 1 mg/L increase in hs-CRP was independently predictive of higher all-cause mortality (hazard ratio [HR], 1.02; 95% CI, 1.01 to 1.04; P = 0.002) and cardiovascular mortality (HR, 1.03; 95% CI, 1.01 to 1.05; P = 0.001) in peritoneal dialysis patients. Other significant predictors for all-cause mortality included age (HR, 1.07; 95% CI, 1.04 to 1.10), gender (HR, 0.49; 95% CI, 0.27 to 0.90), atherosclerotic vascular disease (HR, 2.65; 95% CI, 1.46 to 4.80), left ventricular mass index (HR, 1.01; 95% CI, 1.00 to 1.01) and residual GFR (HR, 0.53; 95% CI, 0.38 to 0.75). Age (HR, 1.06; 95% CI, 1.02 to 1.10), history of heart failure (HR, 3.31; 95% CI, 1.36 to 8.08), atherosclerotic vascular disease (HR, 3.20; 95% CI, 1.43 to 7.13), and residual GFR (HR, 0.57; 95% CI, 0.38 to 0.86) were also independently predictive of cardiovascular mortality. In conclusion, a single, random hs-CRP level has significant and independent prognostic value in PD patients.

The dynamics of the nucleolus.

The nucleolus is the most prominent structure within the eukaryotic cell nucleus and is the site where ribosomal RNAs (5.8S, 18S, and 28S) are transcribed, processed, and assembled with ribosomal proteins to form ribosomal subunits. A role in ribosome biogenesis alone, however, does not account for the specific nucleolar localizations of tumor suppressor proteins, cell cycle regulator factors, and viral proteins. Certain proteins have also been shown to accumulate in the nucleolus only under specific metabolic conditions or at specific cell cycle stages. The use of green fluorescent protein fusions has recently revealed that several proteins localize to distinct subnucleolar compartments via specific targeting pathways. Meanwhile, photobleaching analyses indicate that most nucleolar proteins studied are continually exchanging between the nucleoplasm and the nucleolus. Proteomic studies have also highlighted the dynamic nature of the nucleolar proteome. Both protein composition and the morphology of subnucleolar compartments can change in response to alterations in the levels of transcription and phosphorylation and during mitosis. This review focuses on nucleolar dynamics, studied at each of the single protein, collective proteome, and subnucleolar organization levels.

Large-scale prediction of phenotype: concept.

The capability to gather organism wide data has far outstripped the ability to understand it. Transforming large-scale data into a "better" cell requires tools that integrate physiology with its environment. One such tool is large-scale mathematical models that marry stoichiometry and kinetics with metabolic regulation and control. It is straightforward to determine stoichiometry (at least for central pathways), and kinetics can be roughly approximated where need be. However, the molecular details of the "metabolic wiring" managing the cell are often missing. Presented here is a surrogate for these missing details based on a simple premise; over evolutionary time, biological systems have developed objective-based programs that frugally manage gene expression and enzyme activity. Mathematically, this notion can be represented as sets of nonlinear control or "management" problems which, when solved in parallel with the model balances, offer a prediction of how gene expression and enzyme activity are modulated, in the absence of specific mechanistic details. We present a model of Escherichia coli central carbon metabolism, describing batch aerobic growth on glucose, in which transcription, translation, and activity of the gene products of 45 genes is "managed" using this approach. The model consists of 122 species (metabolites, enzymes, mRNA pools, and biomass) and describes 46 reactions (17 reversible). The model is identified (kinetic parameters as well as management structure) from metabolic flux ratio (METAFoR) analysis and physiological measurements. Simulations of a pyruvate kinase knockout strain are compared with experiments and it is shown the model is capable of accurately capturing the metabolic reprogramming resulting from the deletion. Analysis of the mRNA expression pattern, translational pattern and enzyme activity pattern of the wild-type versus mutant indicates a combination of expression and specific activity shifts are responsible for observed differences. While being only a first step toward large-scale physiological modeling, this work is important in two ways. First, it strengthens the hypothesis that unknown mechanism can be reasonably approximated using objective-based management criteria. Second, it provides a dynamic means to couple large-scale analysis technologies with physiology at the single-gene, single-protein level.

Effect of halothane on human skeletal muscle sarcoplasmic reticulum calcium-release channel.

In order to gain an understanding of how anesthetics alter muscle function, we have utilized the planar lipid bilayer technique for recording the conductance and gating properties of a calcium-release channel molecule derived from native skeletal muscle sarcoplasmic reticulum membranes. The incorporation of this protein molecule into an artificial membrane simplifies investigation, limiting anesthetic action to the bilayer itself and/or the single protein molecule. Thus, any effect the anesthetic has on the protein’s conductance and gating functions will be a consequence of the anesthetic’s action directly on the protein and/or on the lipid bilayer in such a way as to alter the protein’s function. Our attempts to understand a piece of the puzzle of anesthetic action on skeletal muscle do, in fact, begin at the molecular, single protein level of attack and even so, this experimental model is far from simple, and the possible ways by which volatile anesthetics could alter its function represent a challenge. Our piece of the puzzle involves a large protein molecule located in the structure bridging the transverse tubule and the terminal cisternae of the sarcoplasmic reticulum (see Chapter 1, figure 1). This protein, sometimes referred to as the ryanodine receptor protein, is thought to play a significant role in excitation-contraction coupling of skeletal muscle. In our present studies, we have incorporated this protein into a planar lipid bilayer and have recorded and measured its properties as a cation-conducting channel. These studies parallel those on the pharmacogenetic disease malignant hyperthermia (MH) and illustrate how such a disease may lead to a better understanding of normal muscle response to volatile anesthetics.