Modeling of Microvascular Permeability Changes after Electroporation

Vascular endothelium selectively controls the transport of plasma contents across the blood vessel wall. The principal objective of our preliminary study was to quantify the electroporation-induced increase in permeability of blood vessel wall for macromolecules, which do not normally extravasate from blood into skin interstitium in homeostatic conditions. Our study combines mathematical modeling (by employing pharmacokinetic and finite element modeling approach) with in vivo measurements (by intravital fluorescence microscopy). Extravasation of fluorescently labeled dextran molecules of two different sizes (70 kDa and 2000 kDa) following the application of electroporation pulses was investigated in order to simulate extravasation of therapeutic macromolecules with molecular weights comparable to molecular weight of particles such as antibodies and plasmid DNA. The increase in blood vessel permeability due to electroporation and corresponding transvascular transport was quantified by calculating the apparent diffusion coefficients for skin microvessel wall (D [μm²/s]) for both molecular sizes. The calculated apparent diffusion coefficients were D = 0.0086 μm²/s and D = 0.0045 μm²/s for 70 kDa and 2000 kDa dextran molecules, respectively. The results of our preliminary study have important implications in development of realistic mathematical models for prediction of extravasation and delivery of large therapeutic molecules to target tissues by means of electroporation.     

Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model

NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling.     

Intramolecular Interaction Influences Binding of the Flax L5 and L6 Resistance Proteins to their AvrL567 Ligands

L locus resistance (R) proteins are nucleotide binding (NB-ARC) leucine-rich repeat (LRR) proteins from flax (Linum usitatissimum) that provide race-specific resistance to the causal agent of flax rust disease, Melampsora lini. L5 and L6 are two alleles of the L locus that directly recognize variants of the fungal effector AvrL567. In this study, we have investigated the molecular details of this recognition by site-directed mutagenesis of AvrL567 and construction of chimeric L proteins. Single, double and triple mutations of polymorphic residues in a variety of AvrL567 variants showed additive effects on recognition strength, suggesting that multiple contact points are involved in recognition. Domain-swap experiments between L5 and L6 show that specificity differences are determined by their corresponding LRR regions. Most positively selected amino acid sites occur in the N- and C-terminal LRR units, and polymorphisms in the first seven and last four LRR units contribute to recognition specificity of L5 and L6 respectively. This further confirms that multiple, additive contact points occur between AvrL567 variants and either L5 or L6. However, we also observed that recognition of AvrL567 is affected by co-operative polymorphisms between both adjacent and distant domains of the R protein, including the TIR, ARC and LRR domains, implying that these residues are involved in intramolecular interactions to optimize detection of the pathogen and defense signal activation. We suggest a model where Avr ligand interaction directly competes with intramolecular interactions to cause activation of the R protein.     

A non-uniform sampling approach enables studies of dilute and unstable proteins

NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on sample preparation and the high level of control of sample conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated samples or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform sampling (NUS) allows for signal averaging to be monitored in an iterative manner, enabling efficient use of spectrometer time, ultimately leading to savings in costs associated with instrument and isotope-labelled protein use. The method requires preparation of multiple aliquots of the protein sample that are flash-frozen and thawed just before acquisition of a short NMR experiments carried out while the protein is stable (12 h in the presented case). Non-uniform sampling enables sufficient resolution to be acquired for each short experiment. Identical NMR datasets are acquired and sensitivity is monitored after each co-added spectrum is reconstructed. The procedure is repeated until sufficient signal-to-noise is obtained. We discuss how maximum entropy reconstruction is used to process the data, and propose a variation on the previously described method of automated parameter selection. We conclude that combining NUS with iterative co-addition is a general approach, and particularly powerful when applied to unstable proteins.     

The distribution of different classes of nuclear localization signals (NLSs) in diverse organisms and the utilization of the minor NLS-binding site inplantnuclear import factor importin-α

The specific recognition between the import receptor importin-α and the nuclear localization signals (NLSs) is crucial to ensure the selective transport of cargoes into the nucleus. NLSs contain 1 or 2 clusters of positively charged amino acids, which usually bind to the major (monopartite NLSs) or both minor and major NLS-binding sites (bipartite NLSs). In our recent study, we determined the structure of importin-α1a from rice (Oryza sativa), and made 2 observations that suggest an increased utilization of the minor NLS-binding site in this protein. First, unlike the mammalian protein, both the major and minor NLS-binding sites are auto-inhibited in the unliganded rice protein. Second, we showed that NLSs of the “plant-specific” class preferentially bind to the minor NLS-binding site of rice importin-α. Here, we show that a distinct group of “minor site-specific” NLSs also bind to the minor site of the rice protein. We further show a greater enrichment of proteins containing these “plant-specific” and “minor site-specific” NLSs in the rice proteome. However, the analysis of the distribution of different classes of NLSs in diverse eukaryotes shows that in all organisms, the minor site-specific NLSs are much less prevalent than the classical monopartite and bipartite NLSs.     

Thorough clinical evaluation of skin, as well as oral, genital and anal mucosa is beneficial in lichen planus patients

Lichen planus (LP) is a common mucocutaneous disease of unknown aetiology with various geographical prevalence, may be related to some serious disorders such as squamous cell carcinoma and often remains underdiagnosed. The aim of this retrospective study was to thoroughly determine localization and clinical characteristics of LP lesions in a cohort of 173 Slovenian patients in association to the presence of accompanying symptoms and history of potential stressful events. Isolated cutaneous lesions of LP were found in 56.6% and isolated oral LP in 3.5% of patients. Thirty-four percent presented orocutaneous LP, whereas genitocutaneous LP was noted in 1.2%, orogenito-cutaneous LP in 4% and orogenital LP in 0.5% of patients. Underlying stressful events were noted in 36 out of 137 (26.3%) patients. Despite obviously visible localization of the lesions various medical specialists should be familiar with LP and thoroughly examine the complete skin, as well as oral, genital and anal mucosa in each LP patient to avoid a delay in diagnosing this disease and possibly disclose a much serious underlying condition. Psychological support should be offered, if needed.     

A molecular mechanism for bacterial susceptibility to zinc

Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumoniae extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a ΔpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response.     

Biological Properties of Melanoma and Endothelial Cells after Plasmid AMEP Gene Electrotransfer Depend on Integrin Quantity on Cells

The data on the biological responsiveness of melanoma and endothelial cells that are targeted by Antiangiogenic MEtargidin Peptide (AMEP) are limited; therefore, the antiproliferative, antimetastatic and antiangiogenic effects of AMEP were investigated in murine melanoma and human endothelial cells after plasmid AMEP gene electrotransfer into the cells in vitro. Plasmid AMEP, a plasmid coding for the disintegrin domain of metargidin targeting specific integrins, had cytotoxic and antiproliferative effects on murine melanoma and human endothelial cells. Among the metastatic properties of cells, migration, invasion and adhesion were investigated. Plasmid AMEP strongly affected the migration of murine melanoma and human endothelial cell lines and also affected the invasion of highly metastatic murine melanoma B16F10 and human endothelial cell lines. There was no effect on cell adhesion on Matrigel^TM or fibronectin in all cell lines. The antiangiogenic effect was shown with tube formation assay, where human microvascular endothelial cell line (HMEC-1) proved to be more sensitive to plasmid AMEP gene electrotransfer than the human umbilical vein endothelial cell line (HUVEC). The study indicates that antiproliferative and antimetastatic biological responses to gene electrotransfer of plasmid AMEP in murine melanoma cells were dependent on the integrin quantity on melanoma cells and not on the expression level of AMEP. The strong antiangiogenic effect expressed in human endothelial cell lines was only partly dependent on the quantity of integrins and seemed to be plasmid AMEP dose dependent.     

Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88

Background

Higher-order self-assembly of proteins, or “prion-like” polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level.

Results

We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88.

Conclusion

We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital “all-or-none” responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.