Alexander Jurkevich

Ambidextrous binding of cell and membrane bilayers by soluble matrix metalloproteinase-12.

Matrix metalloproteinases (MMPs) regulate tissue remodelling, inflammation and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here we demonstrate the binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labelled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation.

Distribution of the Vasotocin Subtype Four Receptor (VT4R) in the Anterior Pituitary Gland of the Chicken, Gallus gallus, and its Possible Role in the Avian Stress Response

The neurohormone arginine vasotocin (AVT) in non mammalian vertebrates is homologous to arginine vasopressin (AVP) in mammals. Its actions are mediated via G protein‐coupled receptors that belong to the vasotocin/mesotocin family. Because of the known regulatory effects of nonapeptide hormones on anterior pituitary functions, receptor subtypes in that family have been proposed to be located in anterior pituitary cells. Recently, an avian vasotocin receptor subtype designated VT4R has been cloned, which shares 69% sequence homology with a human vasopressin receptor, the V1aR. In the present study, a polyclonal antibody to the VT4R was developed and validated to confirm its specificity to the VT4R. The antibody was used to test the hypothesis that the VT4R is present in the avian anterior pituitary and is specifically associated with certain cell types, where its expression is modulated by acute stress. Western blotting of membrane protein extracts from pituitary tissue, the use of HeLa cells transfected with the VT4R and peptide competition assays all confirmed the specificity of the antibody to the VT4R. Dual‐labelling immunofluorescence microscopy was utilised to identify pituitary cell types that contained immunoreactive VT4R. The receptor was found to be widely distributed throughout the cephalic lobe but not in the caudal lobe of the anterior pituitary. Immunoreactive VT4R was associated with corticotrophs. Approximately 89% of immunolabelled corticotrophs were shown to contain the VT4R. The immunoreactive VT4R was not found in gonadotrophs, somatotrophs or lactotrophs. To determine a possible functional role of the VT4R and previously characterised VT2R, gene expression levels in the anterior pituitary were determined after acute immobilisation stress by quantitative reverse transcriptase‐polymerase chain reaction. The results showed a significant increase in plasma corticosterone levels (three‐ to four‐fold), a significant reduction of VT4R mRNA and an increase of VT2R mRNA (P < 0.05) in acutely immobilised chicks compared to controls. The data suggest a role of the VT4R in the avian stress response.

Neuroendocrine regulation of stress in birds with an emphasis on vasotocin receptors (VTRs)

The neuroendocrine stress response of vertebrates, particularly mammals, comprises at least two types of neuropeptide containing neurons, corticotropin-releasing hormone (CRH) and vasopressin (VP) neurons, and four receptors [CRH receptor one (CRH-R1) and two (CRH-R2) and VP receptor 1a (V1aR) and 1b (V1bR)]. The avian neuropeptide CRH, a 41-amino acid peptide, has been shown to have the same amino acid sequence as humans while nonapeptide neurohormone arginine-vasotocin (AVT) is regarded as highly conserved having a single amino acid substitution compared to mammalian arginine vasopressin. Similar to mammals, birds have two receptor subtypes (CRH-R1 and CRH-R2) for CRH, however, four vasotocin receptors have been identified. Less is known about the functions of the four avian vasotocin receptors compared to homologous ones found in mammals and other vertebrate classes. Recently, chicken vasotocin receptor two (VT2R) and four (VT4R) have been characterized utilizing immunocytochemistry and an imposed stress test. The purpose of this review is to present evidence that the VT2R and VT4R are involved in the avian stress response and that the cephalic lobe of the anterior pituitary appears specialized for this function as it contains the major population of corticotropes and necessary neuroendocrine receptors to respond to stressors impacting avian species.

Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors.

Matrix metalloproteinase-7 (MMP-7) sheds signaling proteins from cell surfaces to activate bacterial killing, wound healing, and tumorigenesis. The mechanism targeting soluble MMP-7 to membranes has been investigated. Nuclear magnetic resonance structures of the zymogen, free and bound to membrane mimics without and with anionic lipid, reveal peripheral binding to bilayers through paramagnetic relaxation enhancements. Addition of cholesterol sulfate partially embeds the protease in the bilayer, restricts its diffusion, and tips the active site away from the bilayer. Its insertion of hydrophobic residues organizes the lipids, pushing the head groups and sterol sulfate outward toward the enzymes positive charge on the periphery of the enlarged interface. Fluorescence probing demonstrates a similar mode of binding to plasma membranes and internalized vesicles of colon cancer cells. Binding of bilayered micelles induces allosteric activation and conformational change in the auto-inhibitory peptide and the adjacent scissile site, illustrating a potential intermediate in the activation of the zymogen.

Distribution of the vasotocin type 4 receptor throughout the brain of the chicken, Gallus gallus.

The vasopressin 1a receptor (V1aR) has been shown to have a wide distribution throughout the mammalian brain and pituitary gland and mediates a number of physiological functions as well as social behavior following the binding of its agonist, vasopressin. The avian receptor homologous to the V1aR is the vasotocin 4 receptor (VT4R). Its mRNA distribution has been documented in brain regions of two species of songbird; however, its complete protein distribution in the brain has not been published to date for any avian species. The present work utilizes an antibody made to a sequence of the chicken VT4R to map its distribution from the olfactory bulbs to the caudal end of the brainstem in Gallus gallus. Unexpectedly, immunoreactivity (ir) for the VT4R was found not only in neurons but also in glia located in 10 circumventricular organs (CVOs), olfactory bulbs, hippocampus, and septum. Use of a second antibody made against vimentin provided evidence that some dual‐labeled glial cells were tanycytes and radial glia. Additionally, the VT4R was identified in nuclei related to motor function, including the oculomotor complex and motor nucleus of the fourth, fifth, sixth, seventh, tenth, and twelfth cranial nerves. Possible functions for the VT4R are suggested that should have relevance not only to avian species but to other vertebrates because most classes, except for mammals, use vasotocin as the natural ligand for that receptor. J. Comp. Neurol. 523:335–358, 2015.

Anatomical and functional implications of CRH neurons in a septal nucleus of the avian brain: An emphasis on glial-neuronal interaction via V1a receptors in vitro

Previously, we showed that corticotrophin‐releasing hormone immunoreactive (CRH‐IR) neurones in a septal structure are associated with stress and the hypothalamic‐pituitary‐adrenal axis in birds. In the present study, we focused upon CRH‐IR neurones located within the septal structure called the nucleus of the hippocampal commissure (NHpC). Immunocytochemical and gene expression analyses were used to identify the anatomical and functional characteristics of cells within the NHpC. A comparative morphometry analysis showed that CRH‐IR neurones in the NHpC were significantly larger than CRH‐IR parvocellular neurones in the paraventricular nucleus of the hypothalamus (PVN) and lateral bed nucleus of the stria terminalis. Furthermore, these large neurones in the NHpC usually have more than two processes, showing characteristics of multipolar neurones. Utilisation of an organotypic slice culture method enabled testing of how CRH‐IR neurones could be regulated within the NHpC. Similar to the PVN, CRH mRNA levels in the NHpC were increased following forskolin treatment. However, dexamethasone decreased forskolin‐induced CRH gene expression only in the PVN and not in the NHpC, indicating differential inhibitory mechanisms in the PVN and the NHpC of the avian brain. Moreover, immunocytochemical evidence also showed that CRH‐IR neurones reside in the NHpC along with the vasotocinergic system, comprising arginine vasotocin (AVT) nerve terminals and immunoreactive vasotocin V1a receptors (V1aR) in glia. Hence, we hypothesised that AVT acts as a neuromodulator within the NHpC to modulate activity of CRH neurones via glial V1aR. Gene expression analysis of cultured slices revealed that AVT treatment increased CRH mRNA levels, whereas a combination of AVT and a V1aR antagonist treatment decreased CRH mRNA expression. Furthermore, an attempt to identify an intercellular mechanism in glial‐neuronal communication in the NHpC revealed that brain‐derived neurotrophic factor (BDNF) and its receptor (TrkB) could be involved in the signalling mechanism. Immunocytochemical results further showed that both BDNF and TrkB receptors were found in glia of the NHpC. Interestingly, in cultured brain slices containing the NHpC, the use of a selective TrkB antagonist decreased the AVT‐induced increase in CRH gene expression levels. The results from the present study collectively suggest that CRH neuronal activity is modulated by AVT via V1aR involving BDNF and TrkB glia in the NHpC.

Peripheral membrane associations of matrix metalloproteinases

Water soluble matrix metalloproteinases (MMPs) have been regarded as diffusing freely in the extracellular matrix. Yet multiple MMPs are also observed at cell surfaces. Their membrane-proximal activities include sheddase activities, collagenolysis, bacterial killing, and intracellular trafficking reaching as far as the nucleus. The catalytic domains of MMP-7 and MMP-12 bind bilayers peripherally, each in two different orientations, by presenting positive charges and a few hydrophobic groups to the surface. Related peripheral membrane associations are predicted for other soluble MMPs. The peripheral membrane associations may support pericellular proteolysis and endocytosis. The isolated soluble domains of MT1-MMP can also associate with membranes. NMR assays suggest transient association of the hemopexin-like domains of MT1-MMP and MMP-12 with lipid bilayers. Peripheral association of soluble MMP domains with bilayers or heparin sulfate proteoglycans probably concentrates them near the membrane. This could increase the probability of forming complexes with membrane-associated proteins, such as those targeted for proteolysis. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death

Atherosclerotic plaque destabilization is the major determinant of most acute coronary events. Smooth muscle cell (SMC) death contributes to plaque destabilization. Here, we describe a novel antiapoptotic mechanism in vascular SMCs that involves interaction of nuclear glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) with apurinic/apyrimidinic endonuclease 1 (Ape1), the major oxidized DNA repair enzyme. GAPDH down‐regulation potentiated H2O2‐induced DNA damage and SMC apoptosis. Conversely, GAPDH overexpression decreased DNA damage and protected SMCs against apoptosis. Ape1 down‐regulation reversed the resistance of GAPDH‐overexpressing cells to DNA damage and apoptosis, which indicated that Ape1 is indispensable for GAPDH‐dependent protective effects. GAPDH bound Ape1 in the SMC nucleus, and blocking (or oxidation) of GAPDH active site cysteines suppressed GAPDH/Ape1 interaction and potentiated apoptosis. GAPDH upregulated Ape1 via a transcription factor homeobox protein Hox‐A5–dependent mechanism. GAPDH levels were reduced in atherosclerotic plaque SMCs, and this effect correlated with oxidative stress and SMC apoptosis. Thus, we demonstrated that nuclear GAPDH/Ape1 interaction preserved Ape1 activity, reduced DNA damage, and prevented SMC apoptosis. Suppression of SMC apoptosis by maintenance of nuclear GAPDH/Ape1 interactions may be a novel therapy to increase atherosclerotic plaque stability.—Hou, X., Snarski, P., Higashi, Y., Yoshida, T., Jurkevich, A., Delafontaine, P., Sukhanov, S. Nuclear complex of glyceraldehyde‐3‐phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant‐induced cell death. FASEB J. 31, 3179–3192 (2017). www.fasebj.org

3D widefield light microscope image reconstruction without dyes

3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample’s 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

Tu1982 Downregulation of Sprouty Is Associated With Inhibition of Epithelial-Mesenchymal Transition in Colorectal Cancer: An Unique Role of Sprouty Proteins

Endosome is a membrane-bounded compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Endosome-associated protein sorting nexin 27 (SNX27) is a unique member of sorting nexin family in possessing a PDZ domain. SNX27-retromer as a major retrieval and recycling hub for a variety of transmembrane proteins, many of which play crucial roles during organism growth and cellular homeostasis. We have previously shown that protein-protein interactions with the PDZ domain play roles