Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.     

Hydrophobic hydration of amphipathic peptides

Biomolecular surfaces and interfaces are commonly found with apolar character. The hydrophobic effect thus plays a crucial role in processes involving association with biomolecular surfaces in the cellular environment. By computer simulation, we compared the hydrogen bonding structures and energetics of the proximal hydration shells of the monomer and dimer from a recent study of an extrinsic membrane peptide, melittin. The two peptides were studied in their amphipathic alpha-helical forms, which possess extended hydrophobic surfaces characterized by different topography. The topography of the peptide-water interface was found to be critical in determining the enthalpic nature of hydrophobic hydration. This topographical dependence has far-reaching implications in the regulation of bioactivities in the presence of amphipathicity. This result also engenders reconsideration of the validity of using free energy parameters that depend solely on the chemical nature of constituent moieties in characterizing hydrophobic hydration of proteins and biomolecules in general.     

RNAs: regulators of bacterial virulence

RNA-based pathways that regulate protein expression are much more widespread than previously thought. Regulatory RNAs, including 5' and 3' untranslated regions next to the coding sequence, cis-acting antisense RNAs and trans-acting small non-coding RNAs, are effective regulatory molecules that can influence protein expression and function in response to external cues such as temperature, pH and levels of metabolites. This Review discusses the mechanisms by which these regulatory RNAs, together with accessory proteins such as RNases, control the fate of mRNAs and proteins and how this regulation influences virulence in pathogenic bacteria.     

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.     

Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular free calcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulin-dependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.     

The myotubularin family: novel phosphoinositide regulators

Phosphatidylinositol 3-phosphate [PtdIns(3)P] acts as a second messenger via the recruitment of diverse signalling proteins to various cellular compartments. Recent advances have highlighted the association of human diseases with mutations in phosphatases that regulate PtdIns(3)P levels. Myotubularin, the gene mutated in myotubular myopathy, functions as a lipid phosphatase with specificity for PtdIns(3)P. It is now apparent that there is an increasing family of proteins that are defined by their significant homology with myotubularin. The myotubularin-related gene family includes proteins that exhibit a lipid phosphatase catalytic motif, those that contain mutations of the critical catalytic residues, and at least one protein that functions as an adapter subunit for PtdIns(3)P-3-phosphatase activity. The present challenge is to understand how deregulation of phosphoinositide metabolism causes human disease.     

Opioid peptides, regulators of acetylcholinesterase activity

Studies have been made on the opioid peptides--enkephalins their fragments, and alpha- and gamma-endorphins with concentrations of 10(-8)-10(-4) M, on acetylcholinesterase of human blood erythrocytes. It was found out that these peptides, which were fragments of one propeptide beta-LPH were reversible effectors of acetylcholinesterase. Enkephalins and a number of their fragments were noncompetitive inhibitors. It was shown that natural pentapeptide has the highest inhibitor activity; decreasing of inhibitor activity or the absence of it was a result of pentapeptide molecules degradation. Short endorphins were noncompetitive activators of acetylcholinesterase.     

Effect of regulators on bacterial autolysis

The aim of this work was to study the effect of autolysis regulators (the fraction of microbial teichoic acids) on the rate of autolysis and the activity of bacterial extracellular lytic enzymes. The regulators of autolysis isolated from 23 cultures belonging to 10 microbial species regulated the rate of autolysis in Bacillus, E. coli and Streptococcus lactis. The regulators either activated or inhibited autolysis depending on the substrate (of a bacterium to be subjected to autolysis). The quantitative dependence of the autolysis rate on the regulator concentration was specific for each pair 'regulator--substrate'. The regulatory properties of the fraction of teichoic acids varied depending on the age of a culture from which they had been isolated. The regulators of autolysis, with an exception of the preparation from E. coli, inhibited the activity of B. subtilis extracellular lytic enzymes in the course of their action on E. coli cells. The possibility for using the regulators of autolysis in microbiological processes is discussed.     

A novel technique to study pore-forming peptides in a natural membrane

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na⁺:Ca²⁺,K⁺ exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K⁺, Na⁺ or Ca²⁺), the OS membrane resistance (R _m ) was typically larger than 1 GΩ in the presence of 1 mM external Ca²⁺. Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in ∼50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.     

Amphibian glucagon family peptides: potent metabolic regulators in fish hepatocytes

Peptides analogous to glucagon-like peptide-1 (GLP-1) have been isolated from amphibian pancreas and intestine, and their amino acid sequences and cDNA structures elucidated. Just like their mammalian counterpart, these peptides are potent insulinotropins in mammalian pancreatic cells. We show here that these peptides also exert strong glycogenolytic actions when applied to dispersed fish hepatocytes. We compared the potencies of three synthetic GLP-1s from Xenopus laevis and two native GLP-1s from Bufo marinus in the activation of glycogenolysis in the hepatocytes of a marine rockfish (Sebastes caurinus) and two freshwater catfish (Ameiurus nebulosus and A. melas), and demonstrated their effectiveness in increasing the degree of phosphorylation of glycogen phosphorylase. We also compared the glycogenolytic potency of the peptides with those of human GLP-1 and glucagons from human and B. marinus. Sensitivity to these peptides is species-specific, with the rockfish responding at lower concentrations to GLP-1s and the two catfish reacting better to glucagons. However, the relative potency of the amphibian GLP-1s and glucagons is similar in the three species. Xenopus GLP-1C (xGLP-1C) is consistently more potent than xGLP-1B, while xGLP-1A displays the smallest activation of glycogenolysis. Similarly, Bufo GLP-1(32)-the peptide with the highest amino acid sequence identity to xGLP-1C-always shows a higher potency than Bufo GLP-1(37), which is closely related to xGLP-1B. The relative hierarchy of these glycogenolytic GLP-1s differs from their ranking as insulinotropins in mammalian beta-cells.In the rockfish system, Bufo glucagon-36, a C-terminally extended glucagon, is more potent than the shorter bovine glucagon and Bufo glucagon-29 in the activation of glycogenolysis; when tested in A. nebulosus hepatocytes, bovine and amphibian glucagons are equipotent. Amphibian GLP-1s and glucagons activate glycogenolysis in fish hepatocytes through increased phosphorylation of glycogen phosphorylase, implying involvement of the adenylyl cyclase/protein kinase A system in signal transduction. We conclude that the broad physiological effectiveness of GLP-1 has been retained throughout vertebrate evolution, and that both insulinotropic activity and glycogenolytic actions belong to the repertoire of GLP-1.     

Phosphoinositides: regulators of membrane traffic and protein function

Phosphoinositides serve as important spatio-temporal regulators of intracellular trafficking and cell signalling events. In addition to their recognition by specific phosphoinositide binding domains present within cytoplasmic adaptor proteins or membrane integral channels and transporters phosphoinositides may affect membrane transport by eliciting conformational changes within proteins or by regulating enzymatic activities. During adaptor-mediated membrane traffic phosphoinositides form part of coincidence detection systems that aid in targeting pools of specific phosphoinositides to select intracellular transport pathways. In this review, we discuss potential mechanisms for conferring selectivity onto the phosphoinositide code as well as possible avenues for future research.     

Hydrophobic pockets at the membrane interface: an original mechanism for membrane protein interactions

The effect of partial digestion by trypsin and GluC protease on the association of the membrane polypeptides of LH1 from Rhodospirillum (Rsp.) rubrum was studied. Trypsin and GluC protease treatments of LH1 result in the cleavage of the first three amino acids from the alpha polypeptide and of the first 18 amino acids from the beta polypeptide, respectively, without any noticeable reorganization of their secondary structure, as measured by attenuated total reflectance Fourier transform IR spectroscopy. However, the enthalpy variation accompanying dimer formation was dramatically reduced by the protease attacks by as much as 80%. Our results show that the alphabeta heterodimer is mainly stabilized by hydrophobic interactions which involve the amino-terminal extensions of the participating polypeptides. Using the close homology between the polypeptides of Rsp. rubrum LH1 and that of Rsp. molischianum LH2, whose structure is known, a structural model for these "hydrophobic pockets" lying close to the membrane interface is proposed.     

Determination of bacterial rod shape by a novel cytoskeletal membrane protein

Cell shape is critical for growth, and some genes are involved in bacterial cell morphogenesis. Here, we report a novel gene, rodZ, required for the determination of rod shape in Escherichia coli. Cells lacking rodZ no longer had rod shape but rather were round or oval. These round cells were smaller than known round mutant cells, including mreB and pbpA mutants; both are known to lose rod shape. Morphogenesis from rod cells to round cells and vice versa, caused by depletion and overproduction of RodZ, respectively, revealed that RodZ could regulate the length of the long axis of the cell. RodZ is a membrane protein with bitopic topology such that the N‐terminal region including a helix‐turn‐helix motif is in the cytoplasm, whereas the C‐terminal region is exposed in the periplasm. GFP–RodZ forms spirals along the lateral axis of the cell beneath the cell membrane, similar to the MreB bacterial actin. Thus, RodZ may mediate spatial information from cytoskeletal proteins in the cytoplasm to a peptidoglycan synthesis machinery in the periplasm.     

Composition of the regulators of bacterial autolysis

This work was aimed at studying the composition of agents regulating bacterial autolysis and isolated from the lysate of Bacillus subtilis 402, B. subtilis R2 and Micrococcus lysodeikticus biomass by extraction with 5% TCA followed by precipitation from the extract with 5 volumes of isopropanol. Fractions activating bacterial autolysis and fractions inhibiting it were found in all of the preparations after separation on Acrylex P-60. Fractions with a molecular mass below 12,600 D activated the autolysis whereas fractions with a molecular mass above 18,400 D inhibited it. The activity of fractions inhibiting the autolysis decreased while that of fractions activating the autolysis increased in the regulating agents isolated from B. subtilis cultures with the aging of the latter. The capability of the fractions to activate the autolysis correlated with the content of amino groups and phosphate in them whereas the capacity to inhibit the autolysis correlated with the content of reducing sugars in the fractions. The preparation of the fraction which activated the autolysis from B. subtilis R2 contained 18 amino acids with the predominance of alanine, glutamic acid, lysine and phenylalanine. Apparently, the regulating properties of the preparations are created with the aid of teichoic acids as well as peptidoglycan and protein fragments associated with the acids.     

Polycationic peptides as nonhormonal regulators of chemosignal systems

This review summarizes and analyzes both literature data and results of our own studies on molecular mechanisms of action of natural and artificially created polycationic peptides on functional activity of heterotrimeric G-protein-coupled signal systems. There are considered peptide toxins from insect venom, synthetic peptides that are derivatives of cytoplasmic loops of receptors of the serpentine type as well as artificially created peptides with linear, branched, and dendrimeric structures. Action of most of these peptides on activity of G-proteins is highly selective and these themselves are able to mimic the hormone-activated receptor to be thereby non-hormonal regulators of the signal systems coupled to heterotrimeric G-proteins.     

Proadrenomedullin-derived peptides as autocrine-paracrine regulators of cell growth

Proadrenomedullin (pADM)-derived peptides, adrenomedullin (ADM) and pADM N-terminal 20 peptide (PAMP), are hypotensive peptides, which are expressed, along with their receptors, in several tissues and organs, the function of which they regulate by acting in an autocrine-paracrine manner. Apart from their involvement in the regulation of blood pressure and fluid and electrolyte homeostasis, pADM-derived peptides appear to play a role in the modulation of cell and tissue growth. Evidence has been provided that ADM: 1) favors the remodeling of cardiovascular system under pathological conditions, by exerting an antiapoptotic effect on endothelial cells and an antiproliferogenic and antimigratory action on vascular smooth-muscle cells during neointimal hyperplasia, and by decreasing proliferation and protein synthesis of cardiac myocytes and fibroblasts. These last two effects are mediated by calcitonin gene-related peptide type 1 (CGRP1) receptors coupled to the adenylate cyclase (AC)/protein kinase (PK) A-dependent cascade; 2) inhibits proliferation and enhances apoptosis of kidney mesangial cells, through the modulation of mitogen-activated PK (MAPK) cascades; 3) stimulates proliferation of adrenal zona glomerulosa cells, acting via CGRP1 receptor coupled to the tyrosine kinase-dependent MAPK cascade, thereby possibly being involved in the maintenance and stimulation of adrenal growth; 4) enhances proliferation of skin and mucosa epithelial cells and fibroblasts, by activating CGRP1 receptor coupled to the AC/PKA signaling pathway; and 5) enhances proliferation of several tumor-cell lines through the activation of the AC/PKA cascade, which suggests a potential role for ADM as promoter of neoplastic growth. The growth effects of PAMP have been far less investigated: findings indicate that this peptide, like ADM, enhances adrenal zona glomerulosa-cell proliferation, and, in contrast with ADM, depresses DNA synthesis in some cancer-cell lines. Both pADM-derived peptides are thought to be involved in embryogenesis, such a contention being based on the demonstration of high pADM-gene expression during the crucial phases of organ growth and differentiation.     

Molecular mechanisms that govern the specificity of Sushi peptides for Gram-negative bacterial membrane lipids

Factor C-derived Sushi peptides (S1 and S3) have been shown to bind lipopolysaccharide (LPS) and inhibit the growth of Gram-negative bacteria but do not affect mammalian cells. On the premise that the composition of membrane phospholipids differs between the microbial and human cells, we studied the modes of interaction between S1 and S3 and the bacterial membrane phospholipids, POPG, in comparison to that with the mammalian cell membrane phospholipids, POPC and POPE. S1 exhibits specificity against POPG, suggesting its preference for bacterial anionic phospholipids, regardless of whether the phospholipids form vesicles in a solution or a monolayer on a solid surface. The specificity of the Sushi peptides for POPG is a consequence of the electrostatic and hydrophobic forces. The unsaturated nature of POPG confers fluidity to the lipid layer, and being in the proximity of LPS in the microenvironmental milieu, POPG probably enhances the insertion of the peptide-LPS complex into the bacterial inner membrane. Furthermore, during its interaction with POPG, the S1 peptide underwent a transition from random to alpha-helical coil, while S3 became a mixture of beta-sheet and alpha-helical structures. This differential structural change in the peptides could be responsible for their different modes of disruption of POPG vesicles. Conceivably, the selectivity for POPG spares the mammalian membranes from undesirable effects of antimicrobial peptides, which could be helpful in designing and developing a new generation of antibiotics and in offering some clues about the specific function of Factor C, a LPS biosensor.