FUNDC1 is a novel mitochondrial-associated-membrane (MAM) protein required for hypoxia-induced mitochondrial fission and mitophagy

Mitochondria need to be fragmented prior to engulfment by phagophores, the precursors to autophagosomes. However, how these 2 processes are finely regulated and integrated is poorly understood. We have shown that the outer mitochondrial membrane protein FUNDC1 is a novel mitochondrial-associated membrane (MAM) protein, enriched at the MAM by interacting with the ER resident protein CANX (calnexin) under hypoxia. As mitophagy proceeds, it dissociates from CANX and preferably recruits DNM1L/DRP1 to drive mitochondrial fission in response to hypoxic stress. In addition, knocking down of FUNDC1, DNM1L or CANX in hypoxic cells increases the number of elongated mitochondria and also reduces the colocalization of autophagosome and mitochondria, thus preventing mitophagy. These findings identify FUNDC1 as a molecular hub integrating mitochondrial fission and mitophagy at the MAM in response to hypoxia.     

Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids

Synaptic plasma membranes (SPM) from the brain are known to have specific binding sites for several steroid hormones, but the mechanisms of membrane transduction of steroid signals is not understood. In this study, corticosterone was found to prevent temperature-dependent dissociation of endogenous calmodlin (CaM) from highly purified SPM from rat cerebral cortex. The steroid stabilizes Ca²⁺-dependent membrane binding of endogenous CaM (78% of total CaM), whereas Ca²⁺-independent binding of CaM (the other 22%) is not affected. The stabilization of membrane binding of endogenous CaM by corticosterone is concentration-dependent, with the maximal effect occurring at steroid concentration of 1 M. The EC₅₀ is estimated as 130 nM, which is almost identical to the K_d of specific binding of the steroid to SPM (120 nM) reported previously. The effect in stabilizing membrane binding of CaM is specific to corticosterone and other glucocorticoids (cortisol, dexamethasone and triamcinolone); gonadal steroids (17-estradiol, progesterone and testosterone) are ineffective. Furthermore, corticosterone administration in vivo (2 mg/kg, i.p.) produced a rapid increase of CaM content in SPM, occurring within 5 min after steroid injection and persisting for at least 20 min. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effect of glucocorticoids in promoting membrane binding of CaM may lead to a cascade of consequences in synaptic membrane function.Special issue dedicated to Dr. Sidney Ochs.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Engineering of a linear inactive analog of human β‐defensin 4 to generate peptides with potent antimicrobial activity

Human β‐defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N‐terminus of HBD4. Our results show that l ‐arginine to d ‐arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l ‐arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d ‐arginine. Substitution of cysteine with the hydrophobic helix‐promoting amino acid α‐aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d ‐arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d ‐amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained ⿼50% of the free enzyme activity at 6.4 mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

The effect of oxysterols on the interaction of Alzheimer's amyloid beta with model membranes

The interaction of amyloid beta (Aβ) peptide with cell membranes has been shown to be influenced by Aβ conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aβ-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aβ in membranes and the peptide-induced membrane dynamics in the presence of cholesterol and 7-ketocholesterol (7keto) or 25-hydroxycholesterol (25OH). Our results have indicated that oxysterols rendered membranes more sensitive to Aβ, in contrast to role of cholesterol in inhibiting Aβ/membrane interaction. We have demonstrated that two oxysterols had different impacts owing to distinct positions of the additional oxygen group in their structures. 7keto-containing cell-sized liposomes exhibited a high propensity toward association with Aβ, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aβ (Aβ-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aβ-40). These findings suggest the enhancing effect of oxysterols on interaction of Aβ with membranes and contribute to clarify the harmful impact of cholesterol on Aβ-induced neurotoxicity by means of its oxidation.     

Mechanical properties of lipid bilayers and regulation of mechanosensitive function

Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed.     

Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Placental specializations of the mountain spiny lizard Sceloporus jarrovi

The lizard Sceloporus jarrovi (Phrynosomatidae) is one of the most widely studied viviparous reptiles of North America. Past research has assumed that placentation in this species is relatively simple and functions mainly in gas exchange. Our examination of the late stage placenta via transmission electron microscopy reveals that S. jarrovi has a unique combination of placental characteristics, with unusual specializations for secretion and absorption. In the chorioallantoic placenta, chorionic and uterine tissues are directly apposed through eggshell loss, and their epithelia are greatly attenuated, enhancing gas exchange; this placenta shows evidence of both nutrient transfer and endocrine function. Contrary to past inferences, a yolk sac placenta forms from the avascular omphalopleure and persists through the end of gestation. The uterine epithelium is enlarged and secretory, and the fetal omphalopleure shows branching absorptive channels and other specializations for uptake. Elsewhere, the omphalopleure develops elongated folds that protrude into a coagulum of degenerating shell membrane and other organic material. Uterine tissue in this region shows specializations for absorption. Placental features in S. jarrovi have unexpected functional implications, and challenge assumptions that specializations for nutrient transfer are confined to matrotrophic species. J. Morphol. 271:1153–1175, 2010. © 2010 Wiley‐Liss, Inc.