Counterion-mediated membrane penetration: cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids

Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a water-octanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.     

Anti-dsDNA Antibodies Promote Initiation,and Acquired Loss of Renal Dnase1 Promotes Progression of Lupus Nephritis in Autoimmune (NZBxNZW)F1 Mice

Background

Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease.

Methodology/Principals

In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis.

Findings

Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs.

Significance

These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.     

Synthesis and conformational analysis of carbasugar bioisosteres of α-l-iduronic acid and its methyl glycoside

The synthesis of two novel carbasugar analogues of α-l-iduronic acid is described in which the ring-oxygen is replaced by a methylene group. In analogy with the conformational equilibrium described for α-l-IdopA, the conformation of the carbasugars was investigated by ¹H and ¹³C NMR spectroscopy. Hadamard transform NMR experiments were utilised for rapid acquisition of ¹H,¹³C-HSQC spectra and efficient measurements of heteronuclear long-range coupling constants. Analysis of ¹H NMR chemical shifts and J_H,H coupling constants extracted by a total-lineshape fitting procedure in conjunction with J_H,C coupling constants obtained by three different 2D NMR experiments, viz., ¹H,¹³C-HSQC-HECADE, J-HMBC and IPAP-HSQC-TOCSY-HT, as well as effective proton-proton distances from 1D ¹H,¹H T-ROE and NOE experiments showed that the conformational equilibrium ⁴C₁?²S_5a?¹C₄ is shifted towards ⁴C₁ as the predominant or exclusive conformation. These carbasugar bioisosteres of α-l-iduronic acid do not as monomers show the inherent flexibility that is anticipated to be necessary for biological activity.     

The Sub-Cellular Localization of WRAP53 Has Prognostic Impact in Breast Cancer

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.     

The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.The S100 proteins comprise a multigene family of low-molecular-weight EF-hand calcium binding and zinc binding proteins (5, 13, 16, 24, 33). To date, 19 different S100 proteins have been assigned to this protein family, and they show different degrees of similarity, ranging from 25 to 56% identity at the amino acid level. With S100B, S100P, and S100Z being the exceptions, the majority of the S100 genes are clustered on human chromosome 1q21 (33). Most S100 proteins serve as calcium sensor proteins that, upon activation, regulate the function and/or subcellular distribution of specific target proteins (13, 33, 47), and they are characterized by common structural motifs, including two low-affinity (K_D [equilibrium dissociation constant] of ∼10 μM to 100 μM) helix-loop-helix calcium binding domains (EF hands) that are separated by a hinge region and flanked by amino- and carboxy-terminal domains. The carboxy-terminal domain is variable among S100 proteins, and it typically is the site that is responsible for the selective interaction of each individual S100 protein with specific target proteins (30). S100 proteins are often upregulated in cancers, in inflammation, and in response to cellular stress (14, 16), suggesting that they function in cell responses to stress situations. Consistent with this hypothesis, stress situations were necessary to reveal phenotypes associated with the S100 knockout in mice (11, 14, 33, 56). Moreover, recent observations revealed a new function for the S100 protein family that included their ability to assist and regulate multichaperone complex-ligand interactions (41, 50, 51).One member of the S100 protein family, S100B, has attracted much interest in the past few years because, like other proteins implicated in neurodegeneration (e.g., amyloid, superoxide dismutase, and dual-specificity tyrosine phosphorylation-regulated kinase 1A), its gene is located within a segment of chromosome 21, which is trisomic in Down''s syndrome (DS). Its expression in the brain of mammals coincides with defined periods of central nervous system (CNS) maturation and cell differentiation (43). In oligodendrocyte progenitor cells (OPC), S100B expression is associated with differentiation, and S100B contributes to OPC differentiation in response to demyelinating insult (11). To understand the contribution of S100B to OPC differentiation, we searched for high-affinity S100B target proteins in this cell type by using far-Western analysis. A major and highly specific S100B target protein was identified, the mitochondrial ATAD3A protein.ATAD3A belongs to a new family of eukaryote-specific mitochondrial AAA⁺ ATPase proteins (17). In the human genome, two genes, Atad3A and Atad3B, are located in tandem on chromosome 1p36.33. The Atad3A gene is ubiquitous among multicellular organisms but absent in yeast. The Atad3B gene is specific to the human genome (27). ATAD3A is a mitochondrial protein anchored into the mitochondrial inner membrane (IM) at contact sites with the outer membrane (OM). Thanks to its simultaneous interaction with the two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the inner and outer membranes and controls diverse cell responses ranging from mitochondrial metabolism, cell growth, and mitochondrial fission 20a, 25). The ATAD3A protein has also been identified as a mitochondrial DNA binding protein (23) and as a cell surface antigen in some human tumors (20, 21). The plasma membrane localization of ATAD3A in tumor cells is suggestive that ATAD3A mitochondrial routing can be compromised in pathological situations such as cancer. To understand the functional response resulting from the interaction between S100B and ATAD3A, we first characterized the minimal interaction domain on ATAD3A for S100B binding using thermodynamic studies of wild-type and ATAD3A variants as well as via nuclear magnetic resonance (NMR) spectroscopy techniques. These studies allowed us to further refine the consensus S100B binding motif, which is conserved in several other S100B target proteins, including the p53 protein and several newly discovered target proteins associated with the cell translational machinery. We next analyzed the cellular interaction of S100B with truncated ATAD3A mutants that harbor the S100B binding domain but that are deficient for mitochondrial import. These studies revealed that S100B could assist ATAD3A mutant proteins during cytoplasmic processing by preventing dysfunctional aggregation events. Our results are discussed in light of the possible function of S100B in assisting the cytoplasmic processing of proteins for proper folding and subcellular localization.     

Effects of Endotoxin and Psychological Stress on Redox Physiology,Immunity and Feather Corticosterone in Greenfinches

Assessment of costs accompanying activation of immune system and related neuroendocrine pathways is essential for understanding the selective forces operating on these systems. Here we attempted to detect such costs in terms of disruption to redox balance and interference between different immune system components in captive wild-caught greenfinches (Carduelis chloris). Study birds were subjected to an endotoxin-induced inflammatory challenge and temporary exposure to a psychological stressor (an image of a predator) in a 2*2 factorial experiment. Injection of bacterial endotoxin resulted in up-regulation of two markers of antioxidant protection – erythrocyte glutathione, and plasma oxygen radical absorbance (OXY). These findings suggest that inflammatory responses alter redox homeostasis. However, no effect on markers of oxidative damage to proteins or DNA in erythrocytes could be detected. We found no evidence that the endotoxin injection interfered with antibody production against Brucella abortus antigen or the intensity of chronic coccidiosis. The hypothesis of within-immune system trade-offs as a cost of immunity was thus not supported in our model system. We showed for the first time that administration of endotoxin can reduce the level of corticosterone deposited into feathers. This finding suggests a down-regulation of the corticosterone secretion cascade due to an endotoxin-induced immune response, a phenomenon that has not been reported previously. Exposure to the predator image did not affect any of the measured physiological parameters.     

Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C

Identifying the viral epitopes targeted by broad neutralizing antibodies (NAbs) that sometimes develop in human immunodeficiency virus type 1 (HIV-1)-infected subjects should assist in the design of vaccines to elicit similar responses. Here, we investigated the activities of a panel of 24 broadly neutralizing plasmas from subtype B- and C-infected donors using a series of complementary mapping methods, focusing mostly on JR-FL as a prototype subtype B primary isolate. Adsorption with gp120 immobilized on beads revealed that an often large but variable fraction of plasma neutralization was directed to gp120 and that in some cases, neutralization was largely mediated by CD4 binding site (CD4bs) Abs. The results of a native polyacrylamide gel electrophoresis assay using JR-FL trimers further suggested that half of the subtype B and a smaller fraction of subtype C plasmas contained a significant proportion of NAbs directed to the CD4bs. Anti-gp41 neutralizing activity was detected in several plasmas of both subtypes, but in all but one case, constituted only a minor fraction of the overall neutralization activity. Assessment of the activities of the subtype B plasmas against chimeric HIV-2 viruses bearing various fragments of the membrane proximal external region (MPER) of HIV-1 gp41 revealed mixed patterns, implying that MPER neutralization was not dominated by any single specificity akin to known MPER-specific monoclonal Abs. V3 and 2G12-like NAbs appeared to make little or no contribution to JR-FL neutralization titers. Overall, we observed significant titers of anti-CD4bs NAbs in several plasmas, but approximately two-thirds of the neutralizing activity remained undefined, suggesting the existence of NAbs with specificities unlike any characterized to date.