FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
Safety, efficacy, and molecular mechanism of claudin-1-specific peptides to enhance blood-nerve-barrier permeability
Sauer(*), R. S., Krug(*), S. M., Hackel(*), D., Staat, C., Konasin(*), N., Yang(*), S., Niedermirtl(*), B., Bosten(*), J., Günther, R., Dabrowski, S., Doppler(*), K., Sommer(*), C., Blasig, I. E., Brack(*), A.; Rittner(*), H. L.
J Control Release, 185:88-98
(2014)

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: The blood-nerve barrier consists of the perineurium and endoneurial vessels. The perineurial barrier is composed of a basal membrane and a layer of perineurial cells sealed by tight junction proteins preventing e.g. application of analgesics for selective regional pain control. One of the barrier-sealing proteins in the blood-nerve barrier is claudin-1. Therefore, the claudin-1-peptidomimetics (C1C2), derived from the first extracellular loop (ECL1) on claudin-1 was developed. In this study, we further evaluated the expression of tight junction proteins in the perineurium in Wistar rats and characterized the specificity, in vivo applicability, mechanism of action as well as the biocompatibility of C1C2. In the perineurium, claudin-19, tricellulin and ZO-1, but no claudin-2, 3, 8 and -11 were expressed. C1C2 specifically bound to the ECL1 of claudin-1 and fluorescent 5,6-carboxytetramethylrhodamine-C1C2 was rapidly internalized. Opening the perineurium with C1C2 reduced the mRNA and protein expression of claudin-1 and increased small and macromolecule permeability into the peripheral nerve. Application of C1C2 facilitated regional analgesia using mu-opioid receptor agonists like DAMGO or morphine without motor impairment in naive rats as well as rats with hind paw inflammation. In contrast the control peptide C2C2 derived from ECL1 on claudin-2 did neither open the barrier nor facilitated opioid-mediated regional analgesia. C1C2 delivery was well tolerated and caused no morphological and functional nerve damage. C1C2 effects could be reversed by interference with the wnt-signal-transduction pathway, specifically the homeobox transcription factor cdx2, using a glycogen-synthase-kinase-3 inhibitor. In summary, we describe the composition of and a pathway to open the perineurial barrier employing a peptide to deliver hydrophilic substances to the peripheral nerve.

Interferon-gamma safeguards blood-brain barrier during experimental autoimmune encephalomyelitis
Ni(*), C., Wang(*), C., Zhang(*), J., Qu(*), L., Liu(*), X., Lu(*), Y., Yang(*), W., Deng(*), J., Lorenz, D., Gao(*), P., Meng(*), Q., Yan(*), X., Blasig, I. E.; Qin(*), Z.
The American journal of pathology, 184:3308-3320
(2014)

Tags: Molecular Cell Physiology (Blasig, I.E.), Cellular Imaging (Wiesner)

Abstract: The function of blood-brain barrier is often disrupted during the progression of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the molecular mechanism of blood-brain barrier modulation during neuroinflammation remains unclear. Herein, we show that the expression of interferon-gamma (IFNgamma) receptor on endothelial cells (ECs) protected mice from the brain inflammation during EAE. IFNgamma stabilized the integrity of the cerebral endothelium and prevented the infiltration of leukocytes into the brain. Further analysis revealed that IFNgamma increased the expression of tight junction proteins zonula occludens protein 1 and occludin, as well as membranous distribution of claudin-5, in brain ECs. Silencing claudin-5 abolished the IFNgamma-mediated improvement of EC integrity. Taken together, our results show that IFNgamma, a pleiotropic proinflammatory cytokine, stabilizes blood-brain barrier integrity and, therefore, prevents brain inflammation during EAE.

Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation
Kooij(*), G., Kopplin(*), K., Blasig, R., Stuiver(*), M., Koning(*), N., Goverse(*), G., van der Pol(*), S. M. A., Hof(*), B. V., Gollasch(*), M., Drexhage(*), J. A. R., Reijerkerk(*), A., Meij(*), I. C., Mebius(*), R., Willnow(*), T. E., Müller(*), D., Blasig, I. E.; de Vries(*), H. E.
Acta Neuropathol, 128:267-277
(2014)

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.

Removal of albumin and immunoglobulins from canine cerebrospinal fluid using depletion kits: a feasibility study
Günther, R., Krause, E., Schümann, M., Ausseil(*), J., Heard(*), J. M., Blasig, I. E.; Haseloff, R. F.
Fluids and barriers of the CNS, 11:14
(2014)

Tags: Molecular Cell Physiology (Blasig, I.E.), Mass Spectrometry (Krause, E.)

Abstract: BACKGROUND: Highly abundant proteins in biological fluids such as serum or cerebrospinal fluid (CSF) can hinder the detection of proteins in lower abundance, e.g., potential biomarkers. Commercial products are available for the depletion of albumin and immunoglobulins (Igs), although most of these kits have not been validated for dog samples. The present study therefore examines the use of different types of depletion kits for dog CSF. FINDINGS: Three kits, with different mechanisms for the depletion of albumin and Igs, were tested with dog CSF specimens. One product significantly decreased the amount of albumin; with all kits, IgG was less efficiently removed than albumin. Mass spectrometry of the fractions eluted from the depletion columns revealed considerable co-depletion of other CSF proteins. CONCLUSIONS: A commercially available depletion kit was identified which depletes albumin and (to a lower extent) immunoglobulins from dog CSF. However, the limited efficacy and the concomitant loss of other proteins from the sample should be taken into account when using this product.

Highly conserved cysteines are involved in the oligomerization of occludin-redox dependency of the second extracellular loop
Bellmann, C., Schreivogel, S., Günther, R., Dabrowski, S., Schümann, M., Wolburg(*), H.; Blasig, I. E.
Antioxid Redox Signal, 20:855-867
(2014)

Tags: Molecular Cell Physiology (Blasig, I.E.), Mass Spectrometry (Krause, E.)

Abstract: UNLABELLED: The tight junction (TJ) marker occludin is a 4-transmembrane domain (TMD) protein with unclear physiological and pathological functions, interacting with other TJ proteins. It oligomerizes and is redox sensitive. However, oligomerization sites and mechanisms are unknown. AIMS: To identify hypoxia-sensitive binding sites, we investigated the consequences of amino-acid substitutions of highly conserved cysteines in human occludin, under normal and hypoxic incubations. RESULTS: (i) The extracellular loop 2 (ECL2) showed homophilic trans- and cis-association between opposing cells and along the cell membrane, respectively, caused by a loop properly folded via an intraloop disulfide bridge between the shielded C216 and C237. Hypoxia and reductants prevented the associations. (ii) C82 in TMD1 directly cis-associated without disulfide formation. (iii) C76 in TMD1 and C148 in TMD2 limited the trans-interaction; C76 also limited occludin-related paracellular tightness and changed the strand morphology of claudin-1. (iv) The diminished binding strength found after substituting C82, C216, or C237 was accompanied by increased occludin mobility in the cell membrane. INNOVATION: The data enable the first experimentally proven structural model of occludin and its homophilic interaction sites, in which the ECL2, via intraloop disulfide formation, has a central role in occludin's hypoxia-sensitive oligomerization and to regulate the structure of TJs. CONCLUSION: Our findings support the new concept that occludin acts as a hypoxiasensor and contributes toward regulating the TJ assembly redox dependently. This is of pathogenic relevance for tissue barrier injury with reducing conditions. The ECL2 disulfide might be a model for four TMD proteins in TJs with two conserved cysteines in an ECL.

Structure of a C. perfringens enterotoxin mutant in complex with a modified Claudin-2 extracellular loop 2
Yelland(*), T. S., Naylor(*), C. E., Bagoban(*), T., Savva(*), C. G., Moss(*), D. S., McClane(*), B. A., Blasig, I. E., Popoff(*), M.; Basak(*), A. K.
J Mol Biol, 426:3134-3147
(2014)

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.

Semisynthesis and optimization of G protein-coupled receptor mimics
Abel, S., Geltinger, B., Heinrich, N., Michl, D., Klose, A., Beyermann, M.; Schwarzer(*), D.
J Pept Sci, 20:831-836
(2014)

Tags: Peptide Chemistry (Beyermann)

Abstract: We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs.

Stretch-activation of angiotensin II type 1a receptors contributes to the myogenic response of mouse mesenteric and renal arteries
Schleifenbaum(*), J., Kassmann(*), M., Szijarto(*), I. A., Hercule(*), H. C., Tano(*), J. Y., Weinert, S., Heidenreich, M., Pathan(*), A. R., Anistan(*), Y. M., Alenina(*), N., Rusch(*), N. J., Bader(*), M., Jentsch, T. J.; Gollasch(*), M.
Circ Res, 115:263-272
(2014)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: RATIONALE: Vascular wall stretch is the major stimulus for the myogenic response of small arteries to pressure. The molecular mechanisms are elusive, but recent findings suggest that G protein-coupled receptors can elicit a stretch response. OBJECTIVE: To determine whether angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. METHODS AND RESULTS: We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, transient receptor potential channel 6 (TRPC6) channels, or several subtypes of the voltage-gated K+ (Kv7) gene family (KCNQ3, 4, or 5). We identified a mechanosensing mechanism in isolated mesenteric arteries and in the renal circulation that relies on coupling of the AT1R subtype a to a Gq/11 protein as a critical event to accomplish the myogenic response. Arterial mechanoactivation occurs after pharmacological block of AT1R and in the absence of angiotensinogen or TRPC6 channels. Activation of AT1R subtype a by osmotically induced membrane stretch suppresses an XE991-sensitive Kv channel current in patch-clamped vascular smooth muscle cells, and similar concentrations of XE991 enhance mesenteric and renal myogenic tone. Although XE991-sensitive KCNQ3, 4, and 5 channels are expressed in vascular smooth muscle cells, XE991-sensitive K+ current and myogenic contractions persist in arteries deficient in these channels. CONCLUSIONS: Our results provide definitive evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand-independent, mechanoactivation of AT1R subtype a. The AT1R subtype a signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4, or 5 to enact vascular smooth muscle cell activation and elevated vascular resistance.

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding
Christian(*), F., Krause, E., Houslay(*), M. D.; Baillie(*), G. S.
Bba-Mol Cell Res, 1843:2765-2774
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic-basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by CAMP-dependent protein kinase (PKA). Such phosphoiylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein. (C) 2014 Elsevier B.V. All rights reserved.

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding
Christian(*), F., Krause, E., Houslay(*), M. D.; Baillie(*), G. S.
Biochim Biophys Acta, 1843:2765-2774
(2014)

Tags: Mass Spectrometry (Krause, E.)

Abstract: p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic-basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by cAMP-dependent protein kinase (PKA). Such phosphorylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
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