Mip Protein Research Articles

A new ELISA method for serological diagnosis of Legionella pneumophila: use of five purified proteins, FLA, MOMP, MIP, IP, and PILE, as diagnostic antigen.

Legionella pneumophila plays an important role in human infection. Commercial ELISA kits commonly used, which take Legionella pneumophila whole-cell protein as the coating antigen, often have cross-reactivity among serogroups or species. In this study, five Legionella pneumophila proteins FLA, MOMP, MIP, IP, and PILE were purified and further applied in serological diagnosis of Legionella pneumophila infections compared with R & D Legionella ELISA kits. The five recombinant plasmids pET-fla, pET-momp, pET-mip, pET-ip, and pET-pile were transformed into E. coli BL21 and then induced them with IPTG. The expression products were analyzed by SDS-PAGE and purified by affinity chromatography. Indirect enzyme-linked immunosorbent assays (ELISAs) were established with the five purified proteins FLA, MOMP, MIP, IP, and PILE altogether as the coating antigen and tested for the presence of IgG, IgM, and IgA antibody independently from 50 positive sera and 40 negative sera, compared with R & D IgG, IgM, and IgA Legionella ELISA kits. The FLA protein about 42 kDa in size, the MOMP protein about 45 kDa, the MIP protein about 40 kDa, the IP protein about 46 kDa, and the PILE protein about 35.7 kDa were separately expressed and purified. Compared with R & D IgG, IgM, and IgA Legionella ELISA kit, the outcome of indirect ELISAs set up with the five purified proteins showed that for IgG the sensitivity was 90.4%, the specificity was 97.4%, the area under ROC curve was 0.939, the kappa value was 0.865, the 95% confidence interval was 0.883 - 0.995. For IgM the sensitivity was 91.8%, the specificity was 95.1%, the area under ROC curve was 0.935, the kappa value was 0.866, the 95% confidence interval was 0.876 - 0.994. For IgA the sensitivity was 93.6%, the specificity was 95.3%, the area under ROC curve was 0.945, the kappa value was 0.889, the 95% confidence interval was 0.890 - 0.999. The proteins FLA, MOMP, MIP, IP, and PILE were successfully expressed and purified, and they seemed to be suitable coating antigens for the serological diagnosis of Legionella pneumophila.

FipB, an essential virulence factor of Francisella tularensis subsp. tularensis, has dual roles in disulfide bond formation.

FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions.

A novel MIP gene mutation analysis in a Chinese family affected with congenital progressive punctate cataract.

Congenital cataracts are one of the leading causes of visual impairment and blindness in children, and genetic factors play an important role in their development. This study aimed to identify the genetic defects associated with autosomal dominant congenital progressive punctate cataracts in a Chinese family and to explore the potential pathogenesis. Detailed family history and clinical data were recorded, and all the family members’ blood samples were collected for DNA extraction. Linkage analysis was performed by microsatellite markers that are associated with punctate cataracts, and logarithm (base 10) of odds (LOD) scores were calculated using the LINKAGE program. Positive two-point LOD scores were obtained at markers D12S1622 (Zmax = 2.71 at θ = 0.0), D12S1724 (Zmax = 2.71 at θ = 0.0), and D12S90 (Zmax = 2.71 at θ = 0.0), which flank the major intrinsic protein of lens fiber (MIP) gene on chromosomal region 12q13. Direct sequencing of the encoding region of the MIP gene revealed a novel mutation (G>D) in exon 4 at nucleotide 644, which caused a substitution of glycine to aspartic acid at codon 215 (p.G215D) for the MIP protein. The mutation cosegregated with all patients with congenital progressive punctate cataracts, but it was absent in the healthy members. Bioinformatics analysis predicted that the mutation affects the function of the MIP protein. The wild type (WT) and G215D mutant of MIP were transfected with green fluorescent protein (GFP) into Hela cells separately, and it was found that the G215D mutant was aberrantly located in the cytoplasm instead of in the plasma membrane. In summary, our study presented genetic and functional evidence linking the new MIP mutation of G215D to autosomal dominant congenital cataracts, which adds to the list of MIP mutations linked to congenital progressive punctate cataracts.

Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs)

Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 μM, 44 ± 3 μM, 17 ± 2 μM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface.

A Structural Biology Approach Enables the Development of Antimicrobials Targeting Bacterial Immunophilins

Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.

MAG2 and three MAG2‐INTERACTING PROTEINs form an ER‐localized complex to facilitate storage protein transport in Arabidopsis thaliana

In Arabidopsis thaliana, MAIGO 2 (MAG2) is involved in protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus via its association with the ER-localized t-SNARE components SYP81/AtUfe1 and SEC20. To characterize the molecular machinery of MAG2-mediated protein transport, we explored MAG2-interacting proteins using transgenic A. thaliana plants expressing TAP-tagged MAG2. We identified three proteins, which were designated as MAG2-INTERACTING PROTEIN 1-3 [MIP1 (At2g32900), MIP2 (At5g24350) and MIP3 (At2g42700)]. Both MIP1 and MAG2 localized to the ER membrane. All of the mag2, mip1, mip2 and mip3 mutants exhibited a defect in storage protein maturation, and developed abnormal storage protein body (MAG body) structures in the ER of seed cells. These observations suggest that MIPs are closely associated with MAG2 and function in protein transport between the ER and Golgi apparatus. MIP1 and MIP2 contain a Zeste-White 10 (ZW10) domain and a Sec39 domain, respectively, but have low sequence identities (21% and 23%) with respective human orthologs. These results suggest that the plant MAG2-MIP1-MIP2 complex is a counterpart of the triple-subunit tethering complexes in yeast (Tip20p-Dsl1p-Sec39p) and humans (RINT1-ZW10-NAG). Surprisingly, the plant complex also contained a fourth member (MIP3) with a Sec1 domain. There have been no previous reports showing that a Sec1-containing protein is a subunit of ER-localized tethering complexes. Our results suggest that MAG2 and the three MIP proteins form a unique complex on the ER that is responsible for efficient transport of seed storage proteins.

Advances in Antioxidative Therapy of Multiple Sclerosis

Multiple sclerosis (MS) is a chronic, multifactorial disease of the central nervous system (CNS), typified by repetitive relapses and/or progression. The conventional treatment options in MS are limited. However, recently several new drugs have been introduced. Oxidative stress is a crucial factor in MS pathogenesis by ameliorating leukocyte migration, contributing to oligodendrocyte damage and axonal injury. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are created in CNS of MS patients mainly by activated macrophages and microglia structures responsible for demyelinisation and axons disruption. Activated microglia secretes different inflammatory and oxidative stress mediators such as cytokines (TNF and IL- 1b and IL- 6) and chemokines (macrophage inflammatory protein MIP- 1a, monocyte chemoattractant protein, MCP- 1 and interferon (IFN) inducible protein IP- 10). The inflammatory state is promoted by that. MS in chronic stages is dominated by neurogenerative processes involving axon and neuron loss probably resulting from oxidative stress and excitotoxicity. Therefore, consideration of the treatment engaging antioxidants and diet supplementation is needed. The present review describes the antioxidative system in CNS and possible antioxidative therapies in MS. Although some exogenous compounds have been proposed as such approach to MS treatment, there is a strong need for further research in this field. Such investigation is required for better understanding of the potential of protective effects of antioxidants in cellular immunology of MS neurodegeneration. Not only would that increase our knowledge about the disease mechanisms but also could help to establish new goals for innovative treatment methods and provide real therapeutic benefits in MS.

Backbone and side-chain 1H, 13C and 15N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii

Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.

Pathogen-free screening of bacteria-specific hybridomas for selecting high-quality monoclonal antibodies against pathogen bacteria as illustrated for Legionella pneumophila

Antibodies are potent biological tools increasingly used as detection, diagnostic and therapeutic reagents. Many technological advances have optimized and facilitated production and screening of monoclonal antibodies. We report here an original method to screen for antibodies targeting biosafety level 2 or 3 pathogens without the fastidious handling inherent to pathogen use. A double ELISA screening was performed using as coated antigen transformed Escherichia coli expressing at its surface a protein specific to the pathogenic bacteria versus control untransformed E. coli. This method was applied to Legionella, using the surface-exposed Mip protein (macrophage infectivity potentiator). This screening proved to be an excellent means of selecting mAbs that bind Legionella pneumophila 1 surface-exposed Mip protein. This method also appears more biologically relevant than screening using the recombinant Mip protein alone and less tedious than a test performed directly on Legionella bacteria. We obtained 21 mAbs that bind strongly to L. pneumophila serogroups 1 to 13, and we validated their use in a rapid ELISA (performed in 4.5h) and an immunochromatographic test (20min).

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.

Enhanced adherence to and invasion of PUVEC and PK-15 cells due to the overexpression of RfaD, ThyA and Mip in the ΔompP2 mutant of Haemophilus parasuis SC096 strain

In Gram-negative bacteria, porins not only contribute to bacterial homeostasis, but also are involved in adherence to and invasion of host cells. Haemophilus parasuis outer membrane protein P2 (OmpP2), a member of the porin family, is an important surface protein involved in serum resistance. To further determine the features of OmpP2, the ability of ompP2 deficient mutant (ΔompP2) of a H. parasuis SC096 strain to interact with porcine umbilicus veins endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15) was evaluated in this study. The ΔompP2 mutant exhibited dramatically increased ability to adhere to and invade PUVEC and PK-15 cells. Conversely, pretreatment of cell lines with purified native OmpP2 porins significantly inhibited adhesion and invasion of the SC096 strain to the both host cells. To explain the unexpected phenomenon, a 2-dimensional gel electrophoresis-based proteomics comparison was performed between the wild-type SC096 and ΔompP2 mutant strains. There were 55 differentially expressed proteins identified from mutant. Among them, three overexpressed proteins of the ADP-l-glycero-d-mannoheptose-6-epimerase RfaD, thymidylate synthase ThyA and putative macrophage infectivity potentiator-related protein Mip were confirmed as molecular targets that modulated adherence and invasion capacities of the ΔompP2 mutant. Collectively, the OmpP2 of the H. parasuis SC096 strain mediated the adherence to and invasion of cells and loss of OmpP2 expression resulted in promoted cell-adherence and invasion properties which were due to the overexpression of RfaD, ThyA and Mip.

Requirement of the CXXC Motif of Novel Francisella Infectivity Potentiator Protein B FipB, and FipA in Virulence of F. tularensis subsp. tularensis

The lipoprotein encoded by the Francisella tularensis subsp. tularensis locus FTT1103 is essential for virulence; an FTT1103 deletion mutant is defective in uptake and intracellular survival, and mice survive high dose challenges of greater than 108 bacteria. This protein has two conserved domains; one is found in a class of virulence proteins called macrophage infectivity potentiator (Mip) proteins, and the other in oxidoreductase Disulfide Bond formation protein A (DsbA)-related proteins. We have designated the protein encoded by FTT1103 as FipB for Francisella infectivity potentiator protein B. The locus FTT1102 (fipA), which is upstream of fipB, also has similarity to same conserved Mip domain. Deletion and site-specific mutants of fipA and fipB were constructed in the Schu S4 strain, and characterized with respect to intracellular replication and in vivo virulence. A nonpolar fipA mutant demonstrated reduced survival in host cells, but was only slightly attenuated in vivo. Although FipB protein was present in a fipA mutant, the abundance of the three isoforms of FipB was altered, suggesting that FipA has a role in post-translational modification of FipB. Similar to many DsbA homologues, FipB contains a cysteine-any amino acid-any amino acid-cysteine (CXXC) motif. This motif was found to be important for FipB's role in virulence; a deletion mutant complemented with a gene encoding a FipB protein in which the first cysteine was changed to an alanine residue (AXXC) failed to restore intracellular survival or in vivo virulence. Complementation with a gene that encoded a CXXA containing FipB protein was significantly defective in intracellular growth; however, only slightly attenuated in vivo.

Effect of interactions between Mip and PrtA on the full extracellular protease activity of Xanthomonas campestris pathovar campestris

Mip (macrophage infectivity potentiator) and Mip-like proteins have been demonstrated to be involved in virulence of several animal pathogens, but as yet none of their native bacterial targets has been identified. Our previous work demonstrated that the Mip-like protein found in the plant pathogen Xanthomonas campestris pv. campestris (Xcc) (hereafter called Mip(Xcc)) is also involved in virulence. Inactivation of the mip(Xcc) gene leads to a significant reduction in exopolysaccharide production and extracellular protease activity via an unknown mechanism. The Xcc genome encodes six extracellular proteases, all of which are secreted via the type II secretion system. The serine protease PrtA makes the largest contribution to Xcc's total extracellular proteolytic activity. In this study, Western blotting analysis demonstrated that Mip(Xcc) was located in the periplasm. Bacterial two-hybrid and far-Western analysis indicated that Mip(Xcc) interacted with PrtA directly. Purified Mip(Xcc) was found to be able to rescue the protease activity of periplasmic proteins extracted from the mip(Xcc) mutant. These findings show that Mip(Xcc) plays a role in the maturation of PrtA, which is the novel native target for at least one Mip or Mip-like protein.

Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration

The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), binds specifically to collagen IV. We identified a surface-exposed Mip-binding sequence in the NC1 domain of human collagen IV α1. The corresponding collagen IV-derived peptide (P290) co-precipitated with Mip and competitively inhibited the Mip-collagen IV binding. Transmigration of Legionella pneumophila across a barrier of NCI-H292 lung epithelial cells and extracellular matrix was efficiently inhibited by P290. This significantly reduced transmigration was comparable to the inefficient transmigration of PPIase-negative Mip mutant or rapamycin-treated L. pneumophila. Based on NMR data and docking studies a model for the mode of interaction of P290 and Mip was developed. The amino acids of the hydrophobic cavity of Mip, D142 and to a lesser extent Y185 were identified to be part of the interaction surface. In the complex structure of Mip(77-213) and P290, both amino acid residues form hydrogen bonds to P290. Utilizing modelling, molecular dynamics (MD) simulations and structural data of human PPIase FKBP12, the most related human orthologue of Mip, we were able to propose optimized P290 variants with increased binding specificity and selectivity for the putative bacterial drug target Mip.

The structure of a Burkholderia pseudomallei immunophilin–inhibitor complex reveals new approaches to antimicrobial development

Mips (macrophage infectivity potentiators) are a subset of immunophilins associated with virulence in a range of micro-organisms. These proteins possess peptidylprolyl isomerase activity and are inhibited by drugs including rapamycin and tacrolimus. We determined the structure of the Mip homologue [BpML1 (Burkholderia pseudomallei Mip-like protein 1)] from the human pathogen and biowarfare threat B. pseudomallei by NMR and X-ray crystallography. The crystal structure suggests that key catalytic residues in the BpML1 active site have unexpected conformational flexibility consistent with a role in catalysis. The structure further revealed BpML1 binding to a helical peptide, in a manner resembling the physiological interaction of human TGFβRI (transforming growth factor β receptor I) with the human immunophilin FKBP12 (FK506-binding protein 12). Furthermore, the structure of BpML1 bound to the class inhibitor cycloheximide N-ethylethanoate showed that this inhibitor mimics such a helical peptide, in contrast with the extended prolyl-peptide mimicking shown by inhibitors such as tacrolimus. We suggest that Mips, and potentially other bacterial immunophilins, participate in protein-protein interactions in addition to their peptidylprolyl isomerase activity, and that some roles of Mip proteins in virulence are independent of their peptidylprolyl isomerase activity.

A Thermosensitive Monolithic Column as an Artificial Antibody for the On‐line Selective Separation of the Protein

The main objective of this study was to develop a new methodology for the preparation of a protein (antigen) that is a molecularly imprinted polymer (MIP, an artificial antibody) modified onto the surface of a silica skeleton in which the resulting stationary phase is thermosensitive. The silica monolithic skeleton with vinyl groups was synthesized in a stainless-steel column by using a mild one-step sol-gel process with two types of precursor: methyltrimethoxysilane (MTMS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS). Subsequently, three types of the thermosensitive protein MIP were anchored onto the surface of the silica skeleton to prepare the MIP monoliths, which were systematically investigated for back pressure and separation ability at different temperatures to establish good imprinting conditions. Under the optimized imprinting conditions, the chromatographic behavior of the thermosensitive MIP monolith exhibited strong retention ability for the lysozyme template (target antigen) in relation to the nonimprinting monolith (NIP monolith). The imprinting factor (IF) for lysozyme reached 3.48 at 20 °C. Moreover, this new type of artificial antibody displayed favorable binding characteristics for lysozyme over competitive proteins and was further evaluated to selectively separate lysozyme in a real sample by using an on-line method. The run-to-run and column-to-column repeatability measurements of the thermosensitive MIP monoliths were also satisfactory.

Muscle-specific inositide phosphatase (MIP/MTMR14) is reduced with age and its loss accelerates skeletal muscle aging process by altering calcium homeostasis

We have recently reported that a novel muscle-specific inositide phosphatase (MIP/MTMR14) plays a critical role in [Ca2+]i homeostasis through dephosphorylation of sn-1-stearoyl-2-arachidonoyl phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2). Loss of function mutations in MIP have been identified in human centronuclear myopathy. We developed a MIP knockout (MIPKO) animal model and found that MIPKO mice were more susceptible to exercise-induced muscle damage, a trademark of muscle functional changes in older subjects. We used wild-type (Wt) mice and MIPKO mice to elucidate the roles of MIP in muscle function during aging. We found MIP mRNA expression, MIP protein levels, and MIP phosphatase activity significantly decreased in old Wt mice. The mature MIPKO mice displayed phenotypes that closely resembled those seen in old Wt mice: i) decreased walking speed, ii) decreased treadmill activity, iii) decreased contractile force, and iv) decreased power generation, classical features of sarcopenia in rodents and humans. Defective Ca2+ homeostasis is also present in mature MIPKO and old Wt mice, suggesting a putative role of MIP in the decline of muscle function during aging. Our studies offer a new avenue for the investigation of MIP roles in skeletal muscle function and as a potential therapeutic target to treat aging sarcopenia.

Co-expression networks: graph properties and topological comparisons

Abstract Motivation: Microarray-based gene expression data have been generated widely to study different biological processes and systems. Gene co-expression networks are often used to extract information about groups of genes that are ‘functionally’ related or co-regulated. However, the structural properties of such co-expression networks have not been rigorously studied and fully compared with known biological networks. In this article, we aim at investigating the structural properties of co-expression networks inferred for the species Saccharomyces Cerevisiae and comparing them with the topological properties of the known, well-established transcriptional network, MIPS physical network and protein–protein interaction (PPI) network of yeast. Results: These topological comparisons indicate that co-expression networks are not distinctly related with either the PPI or the MIPS physical interaction networks, showing important structural differences between them. When focusing on a more literal comparison, vertex by vertex and edge by edge, the conclusion is the same: the fact that two genes exhibit a high gene expression correlation degree does not seem to obviously correlate with the existence of a physical binding between the proteins produced by these genes or the existence of a MIPS physical interaction between the genes. The comparison of the yeast regulatory network with inferred yeast co-expression networks would suggest, however, that they could somehow be related. Conclusions: We conclude that the gene expression-based co-expression networks reflect more on the gene regulatory networks but less on the PPI or MIPS physical interaction networks. Contact: hongzhe@mail.med.upenn.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Usefulness and limitations of dK random graph models to predict interactions and functional homogeneity in biological networks under a pseudo-likelihood parameter estimation approach

BackgroundMany aspects of biological functions can be modeled by biological networks, such as protein interaction networks, metabolic networks, and gene coexpression networks. Studying the statistical properties of these networks in turn allows us to infer biological function. Complex statistical network models can potentially more accurately describe the networks, but it is not clear whether such complex models are better suited to find biologically meaningful subnetworks.ResultsRecent studies have shown that the degree distribution of the nodes is not an adequate statistic in many molecular networks. We sought to extend this statistic with 2nd and 3rd order degree correlations and developed a pseudo-likelihood approach to estimate the parameters. The approach was used to analyze the MIPS and BIOGRID yeast protein interaction networks, and two yeast coexpression networks. We showed that 2nd order degree correlation information gave better predictions of gene interactions in both protein interaction and gene coexpression networks. However, in the biologically important task of predicting functionally homogeneous modules, degree correlation information performs marginally better in the case of the MIPS and BIOGRID protein interaction networks, but worse in the case of gene coexpression networks.ConclusionOur use of dK models showed that incorporation of degree correlations could increase predictive power in some contexts, albeit sometimes marginally, but, in all contexts, the use of third-order degree correlations decreased accuracy. However, it is possible that other parameter estimation methods, such as maximum likelihood, will show the usefulness of incorporating 2nd and 3rd degree correlations in predicting functionally homogeneous modules.

W1215 MDP Stimulates Memory T-Cell Recruitment Via NOD2 Connecting Bacterial Sensing and Adaptive Immunity

BACKGROUND: Macrophage inflammatory protein (Mip)-3α is a CC chemokine attracting memory T lymphocytes and is induced during differentiation of blood monocytes into intestinal macrophages (IMACs) in the intestinal mucosa. It is further released via NFκB activation upon stimulation with proinflammatory cytokines, such as TNF. Bacterial translocation across the epithelial barrier is crucial during the pathogenesis of Crohn's disease (CD). NOD2 is an intracellular sensor of bacterial muramyl dipeptide (MDP) and is able to activate the NF-κB pathway. NOD2 variants have been shown to be the most important susceptibility factor for the development of CD. We have tested the ability of MDP to induce Mip-3α expression and subsequent recruitment of memory T lymphocytes. METHODS: Cells were transfected with vector (pcDNA) to express wtNOD2, SNP13 or empty vector. Cells were stimulated with 0/100/500/1000/2000 ng/ml MDP for 18 hours. Supernatants were collected and Mip-3α was quantified by ELISA and real time-PCR. Functional assays on migration of memory (CD45R0+) T cells were performed in a three dimensional In Vitro differentiation model for IMACs: the multicellular spheroid (MCS) coculture model. Neutralizing antibodies to human Mip-3α (polyclonal, rabbit, acris-antibodies; final concentrations, 15, 1.5, and 0.15 mg/ml) were used for blocking studies. RESULTS: Taqman analysis revealed significantly elevated Mip-3αmRNA expression in HEK293 cells transfected with pcDNA to express NOD2 after stimulation with MDP. Increased Mip-3α secretion was demonstrated by ELISA. MDP induced Mip-3α mRNA and protein secretion in a dose dependent manner. A functional deficit was found after transfection with pcDNA to express SNP13. During the differentiation of monocytes into IMACs Mip-3α was induced in the MCS-In Vitro model during 7 days of coculture. Mip-3α+ IMACs attracted CD45R0+ T cells to migrate into the MCS. CONCLUSION: Mip-3α is induced during differentiation of IMACs. There is evidence for a NOD2 mediated induction of Mip-3α expression followed by subsequent memory T-cell recruitment connecting bacterial sensingwithmemory T-cell functions.