Large‐scale comparison of protein essential dynamics from molecular dynamics simulations and coarse‐grained normal mode analyses

A large‐scale comparison of essential dynamics (ED) modes from molecular dynamic simulations and normal modes from coarse‐grained normal mode methods (CGNM) was performed on a dataset of 335 proteins. As CGNM methods, the elastic network model (ENM) and the rigid cluster normal mode analysis (RCNMA) were used. Low‐frequency normal modes from ENM correlate very well with ED modes in terms of directions of motions and relative amplitudes of motions. Notably, a similar performance was found if normal modes from RCNMA were used, despite a higher level of coarse graining. On average, the space spanned by the first quarter of ENM modes describes 84% of the space spanned by the five ED modes. Furthermore, no prominent differences for ED and CGNM modes among different protein structure classes (CATH classification) were found. This demonstrates the general potential of CGNM approaches for describing intrinsic motions of proteins with little computational cost. For selected cases, CGNM modes were found to be more robust among proteins that have the same topology or are of the same homologous superfamily than ED modes. In view of recent evidence regarding evolutionary conservation of vibrational dynamics, this suggests that ED modes, in some cases, might not be representative of the underlying dynamics that are characteristic of a whole family, probably due to insufficient sampling of some of the family members by MD. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid)

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.     

Identification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> genes modulating the cytokine response of human peripheral blood mononuclear cells

Background  

Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs).     

Comparison of the human and canine cytokines IL-1(alpha/beta) and TNF-alpha to orthologous other mammalians

The cytokines interleukin-1 (IL-1alpha and IL-1beta) and the tumor necrosis factor-alpha (TNF-alpha) both play a major role in the initiation and regulation of inflammation and immunity responses. Polymorphisms within the gene sequences of these cytokines IL-1 and TNF-alpha have been proposed to play an important role in the pathogenesis of certain diseases. Affecting nearly every organ, various diseases, including some cancers, are described to be associated with an increased level of IL-1 and TNF-alpha proteins, for example, solid tumors, hematologic malignancies, malignant histiocytosis, autoimmune disorders, Alzheimer's disease, Parkinson's disease, sepsis, and rheumatoid arthritis. Regarding genetic backgrounds and pathways, numerous canine diseases show close similarities to their human counterparts. As a genetic model, the dog could be used to unravel the genetic mechanisms, for example, in particular the predispositions, the development, and progression of cancer and metabolic diseases. The identity comparison of gene and protein sequences of different species could be used to elucidate the structure and function of the genes and proteins by identifying the evolutionary conserved regions and domains. Herein we analyzed in detail the mRNA and protein structures and identities of the present known mammalian (human, canine, murine, rat, ovine, equine, feline, porcine, and bovine) TNF-alpha, IL-1alpha, and IL-1beta mRNAs and proteins. Additionally, based on the canine genome sequence, we derived in silico the complete mRNA structures of the IL-1alpha and IL-1beta mRNAs.     

The Par-Tiam1 complex controls persistent migration by stabilizing microtubule-dependent front-rear polarity

BACKGROUND: The establishment and maintenance of cell polarity is crucial for many biological functions and is regulated by conserved protein complexes. The Par polarity complex consisting of Par3, Par6, and PKCzeta, in conjunction with Tiam1-mediated Rac signaling, controls apical-basal cell polarity in contacting epithelial cells. Here we tested the hypothesis that the Par complex, in conjunction with Tiam1, controls "front-rear" polarity during the persistent migration of freely migrating keratinocytes. RESULTS: Wild-type (WT) epidermal keratinocytes lacking cell-cell contacts are stably front-rear polarized and migrate persistently. In contrast, Tiam1-deficient (Tiam1 KO) and (si)Par3-depleted keratinocytes are generally unpolarized and migrate randomly because front-rear polarity is short lived. Immunoprecipitation experiments show that in migrating keratinocytes, Tiam1 associates with Par3 and PKCzeta. Moreover, Par3, PKCzeta, and Tiam1 proteins are enriched at the leading edges of polarized keratinocytes. Tiam1 KO keratinocytes are impaired in chemotactic migration toward growth factors, whereaes haptotactic migration is similar to WT. Par3 depletion or the blocking of PKCzeta signaling in WT keratinocytes impairs chemotaxis but has no additional effect on Tiam1 KO cells. The migratory and morphological defects in keratinocytes with impaired Par-Tiam1 function closely resemble cells with pharmacologically destabilized microtubules (MTs). Indeed, MTs in Tiam1 KO keratinocytes and WT cells treated with a PKCzeta inhibitor are unstable, thereby negatively influencing directional but not random migration. CONCLUSIONS: We conclude that the Par-Tiam1 complex stabilizes front-rear polarization of noncontacting migratory cells, thereby stimulating persistent and chemotactic migration, whereas in contacting keratinocytes, the same complex controls the establishment of long-lasting apical-basal polarity. These findings underscore a remarkable flexibility of the Par polarity complex that, depending on the biological context, controls distinct forms of cellular polarity.     

Inter-ring communication allows the GroEL chaperonin complex to distinguish between different substrates

The productive folding of substrate proteins by the GroEL complex of Escherichia coli requires the activity of both the chaperonin rings. These heptameric rings were shown to regulate the chaperonins' affinity for substrates and co-chaperonin via inter-ring communications; however, the molecular details of the interactions are not well understood. We have investigated the effect of substrate binding on inter-ring communications of the chaperonin complex, both the double-ring GroEL as well as the single-ring SR1 chaperonin in complex with four different substrates by using mass spectrometry. This approach shows that whereas SR1 is unable to distinguish between Rubisco, gp23, gp5, and MDH, GroEL shows clear differences upon binding these substrates. The most distinctive binding behavior is observed for Rubisco, which only occupies one GroEL ring. Both bacteriophage capsid proteins (gp23 and gp5) as well as MDH are able to bind to the two GroEL rings simultaneously. Our data suggest that inter-ring communication allows the chaperonin complex to differentiate between substrates. Using collision induced dissociation in the gas phase, differences between the chaperonin(substrate) complexes are observed only when both rings are present. The data indicate that the size of the substrate is an important factor that determines the degree of stabilization of the chaperonin complex.     

CRM1-mediated nuclear export: to the pore and beyond

CRM1 (chromosome region maintenance 1; also referred to as exportin1 or Xpo1) is a member of the importin beta superfamily of nuclear transport receptors, recognizing proteins bearing a leucine-rich nuclear export sequence. CRM1 is the major receptor for the export of proteins out of the nucleus and is also required for transport of many RNAs. Besides its established role in nuclear export, CRM1 is also implicated in various steps during mitosis, widening its functional spectrum within the cell.     

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.