Improved reproducibility of reverse‐phase protein microarrays using array microenvironment normalization

We introduce a novel experimental methodology for the reverse‐phase protein microarray platform which reduces the typical measurement CV as much as 70%. The methodology, referred to as array microenvironment normalization, increases the statistical power of the platform. In the experiment, it enabled the detection of a 1.1‐fold shift in prostate specific antigen concentration using approximately six technical replicates rather than the 37 replicates previously required. The improved reproducibility and statistical power should facilitate clinical implementation of the platform.     

Identification of Novel Serological Biomarkers for Inflammatory Bowel Disease Using Escherichia coli Proteome Chip

Specific antimicrobial antibodies present in the sera of patients with inflammatory bowel disease (IBD) have been proven to be valuable serological biomarkers for diagnosis/prognosis of the disease. Herein we describe the use of a whole Escherichia coli proteome microarray as a novel high throughput proteomics approach to screen and identify new serological biomarkers for IBD. Each protein array, which contains 4,256 E. coli K12 proteins, was screened using individual serum from healthy controls (n = 39) and clinically well characterized patients with IBD (66 Crohn disease (CD) and 29 ulcerative colitis (UC)). Proteins that could be recognized by serum antibodies were visualized and quantified using Cy3-labeled goat anti-human antibodies. Surprisingly significance analysis of microarrays identified a total of 417 E. coli proteins that were differentially recognized by serum antibodies between healthy controls and CD or UC. Among those, 169 proteins were identified as highly immunogenic in healthy controls, 186 proteins were identified as highly immunogenic in CD, and only 19 were identified as highly immunogenic in UC. Using a supervised learning algorithm (k-top scoring pairs), we identified two sets of serum antibodies that were novel biomarkers for specifically distinguishing CD from healthy controls (accuracy, 86 ± 4%; p < 0.01) and CD from UC (accuracy, 80 ± 2%; p < 0.01), respectively. The Set 1 antibodies recognized three pairs of E. coli proteins: Era versus YbaN, YhgN versus FocA, and GabT versus YcdG, and the Set 2 antibodies recognized YidX versus FrvX. The specificity and sensitivity of Set 1 antibodies were 81 ± 5 and 89 ± 3%, respectively, whereas those of Set 2 antibodies were 84 ± 1 and 70 ± 6%, respectively. Serum antibodies identified for distinguishing healthy controls versus UC were only marginal because their accuracy, specificity, and sensitivity were 66 ± 5, 69 ± 5, and 61 ± 7%, respectively (p < 0.04). Taken together, we identified novel sets of serological biomarkers for diagnosis of CD versus healthy control and CD versus UC.Crohn disease (CD)¹ and ulcerative colitis (UC) are chronic, idiopathic, and clinically heterogeneous intestinal disorders collectively known as inflammatory bowel disease (IBD) (1, 2). Although the distinction between UC and CD would seem clear based on the combination of clinical, endoscopic, and radiological criteria, indeterminate colitis is present in up to 10 and 20% of adult and pediatric patients with isolated colitis, respectively (3, 4).Serological testing is a non-invasive method for diagnosing IBD and differentiating UC from CD (5–7). Several serological IBD biomarkers have been identified in the past decade, and some have been used in IBD clinics (5–7) (see the list below). Many of these antibodies are produced on intestinal exposure to normal commensal bacteria in genetically susceptible individuals. Although it is not known whether these antibodies are pathogenic or not, they are specific to patients with either CD or UC and may reflect a dysregulated immune inflammatory response to intestinal bacterial antigens (2, 8–10). Several experimental animal models of IBD have led to the theory that the pathogenesis of IBD is the result of an aberrant immune response to normal commensal bacteria in genetically susceptible individuals (11, 12). In fact, most of the major serological biomarkers being used in IBD clinics are antibodies to microbial antigens, including yeast oligomannose (anti-Saccharomyces cerevisiae (ASCA)), bacterial outer membrane porin C (OmpC), Pseudomonas fluorescens bacterial sequence I2 (anti-I2), and most recently bacterial flagellin (CBir 1) (5–7, 13). All of these antimicrobial antibodies show a preponderance in patients with CD. However, ASCA has been identified in up to 5% of patients with UC (13, 14).In comparison, perinuclear anti-neutrophil cytoplasmic antibody (pANCA) with perinuclear highlighting was first described in 1990. Although generally considered an autoantibody, the specific antigenic stimulation for pANCA production remains unclear. This autoantibody is present in up to 70% of patients with UC and in up to 20% of patients with CD (6, 10). Recently a panel of five new anti-glycan antibodies have been identified, including anti-chitobioside IgA, anti-laminaribioside IgG, anti-mannobioside IgG, and antibodies against two major chemically synthesized (Σ) oligomannose epitopes, Man α-1,3 Man α-1,2 Man (ΣMan3) and Man α-1,3 Man α-1,2 Man α-1,2 Man (ΣMan4) (5, 13, 15). These new biomarkers serve as valuable complimentary tools to the available serological biomarkers mentioned above. Collectively these antibodies are not generally present in either children or adults with non-IBD disease and may represent serological markers of intestinal inflammation specific to UC or CD.Although encouraging, none of the current commercially available biomarker tests/assays, including all of those mentioned above, can be used as stand alone tools in clinics and therefore are only recommended as an adjunct to endoscopy in diagnosis and prognosis of the disease (5, 7, 16). Therefore, additional specific and sensitive IBD biomarkers are needed as are prospective studies to assess the utility of current and newly identified biomarkers (5, 13). Proteomics technologies such as two-dimensional gel electrophoresis, various variations of mass spectrometry, and protein chip (array) technology are now proving to be powerful tools in biomarker discovery and are beginning to be utilized in IBD biomarker discovery (5, 17). These technologies enable robust and/or large scale and high throughput identification and analysis of differential protein expression when comparing disease with control. Blood-based (serum- or plasma-based) proteomics holds particular promises for biomarker discovery of various human diseases such as neurodegenerative diseases and cancers (18–20). Antigen microarrays are also powerful tools that allow high throughput serum analysis of aberrant immune responses in autoimmune diseases (21–23) as well as efficient discovery of biomarkers for infectious pathogens (24). Herein we describe the use of an Escherichia coli proteome microarray to characterize the differential immune response (serum anti-E. coli antibodies) among patients clinically classified as CD, UC, and healthy controls. We hypothesized that novel IBD-specific antimicrobial antibodies, particularly anti-E. coli antibodies, are present in IBD patients and are likely to be identified by screening the sera with E. coli protein arrays.     

Cardiac myocytes and local signaling in nano-domains

It is well known that calcium-induced calcium-release in cardiac myocytes takes place in spatially restricted regions known as dyads, where discrete patches of junctional sarcoplasmic reticulum tightly associate with the t-tubule membrane. The dimensions of a dyad are so small that it contains only a few Ca²⁺ ions at any given time. Ca²⁺ signaling in the dyad is therefore noisy, and dominated by the Brownian motion of Ca²⁺ ions in a potential field. Remarkably, from this complexity emerges the integrated behavior of the myocyte in which, under normal conditions, precise control of Ca²⁺ release and muscle contraction is maintained over the life of the cell. This is but one example of how signal processing within the cardiac myocyte and other cells often occurs in small “nano-domains” where proteins and protein complexes interact at spatial dimensions on the order of ∼1-10 nm and at time-scales on the order of nanoseconds to perform the functions of the cell. In this article, we will review several examples of local signaling in nano-domains, how it contributes to the integrative behavior of the cardiac myocyte, and present computational methods for modeling signal processing within these domains across differing spatio-temporal scales.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Determination of anti-acetylcholine receptor antibodies in myasthenia and its treatment by plasma exchange and immunosuppressants

Recent works have confirmed the auto immune mechanism in myasthenia gravis, that Simpson had hypothetized early as 1960. A post synaptic blockage, of which Ach-R antibodies seem to be chiefly responsible, is now accepted though the exact pathogenesis remains unknown. The immuno assay of these antibodies is still difficult; their blood level evaluates the course of the disease and the effectiveness of the treatment. The withdrawal of the antibodies by plasma exchange (P.E.) and the inhibition of their secretion by immunosuppression (I.S.) lead to remission in many cases. Principle of the immuno assay and results of P.E. and I.S. are reported in 21 cases.     

On the Adjacency Matrix of RyR2 Cluster Structures

In the heart, electrical stimulation of cardiac myocytes increases the open probability of sarcolemmal voltage-sensitive Ca²⁺ channels and flux of Ca²⁺ into the cells. This increases Ca²⁺ binding to ligand-gated channels known as ryanodine receptors (RyR2). Their openings cause cell-wide release of Ca²⁺, which in turn causes muscle contraction and the generation of the mechanical force required to pump blood. In resting myocytes, RyR2s can also open spontaneously giving rise to spatially-confined Ca²⁺ release events known as “sparks.” RyR2s are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. Our recent work has shown that the spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca²⁺ sparks. We showed that the probability of a Ca²⁺ spark occurring when a single RyR2 in the cluster opens spontaneously can be predicted from the precise spatial arrangements of the RyR2s. Thus, “function” follows from “structure.” This probability is related to the maximum eigenvalue (λ ₁) of the adjacency matrix of the RyR2 cluster lattice. In this work, we develop a theoretical framework for understanding this relationship. We present a stochastic contact network model of the Ca²⁺ spark initiation process. We show that λ ₁ determines a stability threshold for the formation of Ca²⁺ sparks in terms of the RyR2 gating transition rates. We recapitulate these results by applying the model to realistic RyR2 cluster structures informed by super-resolution stimulated emission depletion (STED) microscopy. Eigendecomposition of the linearized mean-field contact network model reveals functional subdomains within RyR2 clusters with distinct sensitivities to Ca²⁺. This work provides novel perspectives on the cardiac Ca²⁺ release process and a general method for inferring the functional properties of transmembrane receptor clusters from their structure.     

Radiation stability of proteinase K crystals grown by LB nanotemplate method

A detailed analysis of structural and intensity changes induced by X-ray radiation is presented for two types of proteinase K crystals: crystal grown by classical hanging drop method and those grown by Langmuir–Blodgett (LB) nanotemplate. The comparison of various parameters (e.g. intensity per sigma ratio, unit-cell volume, number of unique reflections, B-factors) and electron density maps as a function of radiation dose, demonstrates that crystals, grown by the LB nanotemplate method, appear to be more resistant against radiation damage than crystals grown by the classical hanging drop method.     

Crystal structure of the UPF2-interacting domain of nonsense-mediated mRNA decay factor UPF1

UPF1 is an essential eukaryotic RNA helicase that plays a key role in various mRNA degradation pathways, notably nonsense-mediated mRNA decay (NMD). In combination with UPF2 and UPF3, it forms part of the surveillance complex that detects mRNAs containing premature stop codons and triggers their degradation in all organisms studied from yeast to human. We describe the 3 A resolution crystal structure of the highly conserved cysteine-histidine-rich domain of human UPF1 and show that it is a unique combination of three zinc-binding motifs arranged into two tandem modules related to the RING-box and U-box domains of ubiquitin ligases. This UPF1 domain interacts with UPF2, and we identified by mutational analysis residues in two distinct conserved surface regions of UPF1 that mediate this interaction. UPF1 residues we identify as important for the interaction with UPF2 are not conserved in UPF1 homologs from certain unicellular parasites that also appear to lack UPF2 in their genomes.     

Initial events in the photocycle of photoactive yellow protein

The light-induced isomerization of a double bond is the key event that allows the conversion of light energy into a structural change in photoactive proteins for many light-mediated biological processes, such as vision, photosynthesis, photomorphogenesis, and photo movement. Cofactors such as retinals, linear tetrapyrroles, and 4-hydroxy-cinnamic acid have been selected by nature that provide the essential double bond to transduce the light signal into a conformational change and eventually, a physiological response. Here we report the first events after light excitation of the latter chromophore, containing a single ethylene double bond, in a low temperature crystallographic study of the photoactive yellow protein. We measured experimental phases to overcome possible model bias, corrected for minimized radiation damage, and measured absorption spectra of crystals to analyze the photoproducts formed. The data show a mechanism for the light activation of photoactive yellow protein, where the energy to drive the remainder of the conformational changes is stored in a slightly strained but fully cis-chromophore configuration. In addition, our data indicate a role for backbone rearrangements during the very early structural events.     

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 A resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.