A stochastic model for then-compartment irreversible system

This paper deals with a stochasticn-compartment irreversible system with a non-homogeneous Poisson input and arbitrary residence time for each of the compartments. Results relating to the number of particles present in each of the compartments as well as the total number of particles present in the system at any time are derived. Further, explicit expressions for the auto covariance function for each compartment and the cross-covariance function between any two compartments with a given time lag are obtained. As a particular case, then-compartment irreversible system is analyzed with homogeneous Poisson input and exponential residence time distribution for each of the compartments. The possible applications of the model are discussed.     

Enzymatic reactions in small spatial volumes: comment on a model of Hess and Mikhailov

Recently Hess and Mikhailov pointed out that in small subcellular compartments diffusion is so fast that mixing is instantaneous on the time scale of many enzymatic reactions. This opens the possibility for synchronizing individual reaction events. To illustrate this fact they discuss as example an irreversible enzymatic reaction with allosteric product activation. Under appropriate conditions their model shows coherent spiking in the number of product molecules, caused by the strong correlation between reaction events. In this model only substrate binding is an indeterministic process, all other subsequent transitions between different enzyme states being deterministic, contrary to real processes. The purpose of the present paper was to investigate this interesting phenomenon by means of a more realistic modification of the original model, with only probabilistic transitions. In an attempt to obtain spiking, which was not observed under these conditions, the model was extended to make a clear distinction between allosteric high and low affinity substrate binding, in contrast to the original model using a product dependent mean binding probability. However no periodic signal was detectable in the indeterministic version of the Hess Mikhailov model or the extended version, either by means of direct visualization or on autocorrelation or Fourier analysis. Reasons why spiking is not observed in indeterministic enzyme models are discussed.     

Oxidative damage to extracellular matrix and its role in human pathologies

The extracellular compartments of most biological tissues are significantly less well protected against oxidative damage than intracellular sites and there is considerable evidence for such compartments being subject to a greater oxidative stress and an altered redox balance. However, with some notable exceptions (e.g., plasma and lung lining fluid) oxidative damage within these compartments has been relatively neglected and is poorly understood. In particular information on the nature and consequences of damage to extracellular matrix is lacking despite the growing realization that changes in matrix structure can play a key role in the regulation of cellular adhesion, proliferation, migration, and cell signaling. Furthermore, the extracellular matrix is widely recognized as being a key site of cytokine and growth factor binding, and modification of matrix structure might be expected to alter such behavior. In this paper we review the potential sources of oxidative matrix damage, the changes that occur in matrix structure, and how this may affect cellular behavior. The role of such damage in the development and progression of inflammatory diseases is discussed.     

Twists and turns of the cation-dependent mannose 6-phosphate receptor. Ligand-bound versus ligand-free receptor.

Mannose 6-phosphate receptors (MPRs) participate in the biogenesis of lysosomes in higher eukaryotes by transporting soluble acid hydrolases from the trans-Golgi network to late endosomal compartments. The receptors release their ligands into the acidic environment of the late endosome and then return to the trans-Golgi network to repeat the process. However, the mechanism that facilitates ligand binding and dissociation upon changes in pH is not known. We report the crystal structure of the extracytoplasmic domain of the homodimeric cation-dependent MPR in a ligand-free form at pH 6.5. A comparison of the ligand-bound and ligand-free structures reveals a significant change in quaternary structure as well as a reorganization of the binding pocket, with the most prominent change being the relocation of a loop (residues Glu(134)-Cys(141)). The movements involved in the bound-to-free transition of the cation-dependent MPR are reminiscent of those of the oxy-to-deoxy hemoglobin transition. These results allow us to propose a mechanism by which the receptor regulates its ligand binding upon changes in pH; the pK(a) of Glu(133) appears to be responsible for ligand release in the acidic environment of the late endosomal compartment, and the pK(a) values of the sugar phosphate and His(105) are accountable for its inability to bind ligand at the cell surface where the pH is about 7.4.     

Protein localization with flexible DNA or RNA

Localization of activity is ubiquitous in life, and also within sub-cellular compartments. Localization provides potential advantages as different proteins involved in the same cellular process may supplement each other on a fast timescale. It might also prevent proteins from being active in other regions of the cell. However localization is at odds with the spreading of unbound molecules by diffusion. We model the cost and gain for specific enzyme activity using localization strategies based on binding to sites of intermediate specificity. While such bindings in themselves decrease the activity of the protein on its target site, they may increase protein activity if stochastic motion allows the acting protein to touch both the intermediate binding site and the specific site simultaneously. We discuss this strategy in view of recent suggestions on long non-coding RNA as a facilitator of localized activity of chromatin modifiers.     

Analysis of cell flow and cell loss following X-irradiation using sequential investigation of the total number of cells in the various parts of the cell cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. The generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G2 blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. The experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

The prediction of protein subcellular localization from sequence: a shortcut to functional genome annotation.

Automated sequence annotation is a major goal of post-genomic era with hundreds of genomes in the databases, from both prokaryotes and eukaryotes. While the number of fully sequenced chromosomes from microbial organisms exponentially increased in the last decade above 600, presently we know the whole DNA content of only 25 eukaryotic organisms, including Homo sapiens. However, the process of genome annotation is far from being completed. This is particularly relevant in eukaryotes, whose cells contain several subcellular compartments, or organelles, enclosed by membranes, where different relevant functions are performed. Translocation across the membrane into the organelles is a highly regulated and complex cellular process. Indeed different proteins and/or protein isoforms, originated from genes by alternative splicing, may be conveyed to different cell compartments, depending on their specific role in the cell. During recent years the prediction of subcellular localization (SL) by computational means has been an active research area. Several methods are presently available based on different notions and addressing different aspects of SL. This review provides a short overview of the most well performing methods described in the literature, highlighting their predictive capabilities and different applications.     

Rabx-5 regulates RAB-5 early endosomal compartments and synaptic vesicles in C. elegans

Early endosomal membrane compartments are required for the formation and recycling of synaptic vesicles, but how these compartments are regulated is incompletely understood. We performed a forward genetic screen in C. elegans for mutations that affect RAB-5 labeled early endosomal compartments in GABAergic motoneurons. Here we report the isolation and characterization of one mutation, rabx-5. The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon. This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5. Synaptic vesicle accumulation is altered in rabx-5 mutants, and synaptic transmission from cholinergic neurons is decreased. Early endosomal membrane compartments show disorganization with ageing and rabx-5 mutant animals age faster. These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.     

Analysis of Cell Flow and Cell Loss Following X-Irradiation Using Sequential Investigation of the Total Number of Cells In the Various Parts of the Cell Cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. the generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G₂ blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. the experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Dissecting physical structure of calreticulin,an intrinsically disordered Ca2+-buffering chaperone from endoplasmic reticulum

Calreticulin (CALR) is a Ca²⁺ binding multifunctional protein that mostly resides in the endoplasmic reticulum (ER) and plays a number of important roles in various physiological and pathological processes. Although the major functions ascribed to CALR are controlling the Ca²⁺ homeostasis in ER and acting as a lectin-like ER chaperon for many glycoproteins, this moonlighting protein can be found in various cellular compartments where it has many non-ER functions. To shed more light on the mechanisms underlying polyfunctionality of this moonlighting protein that can be found in different cellular compartments and that possesses a wide spectrum of unrelated biological activities, being able to interact with Ca²⁺ (and potentially other metal ions), RNA, oligosaccharides, and numerous proteins, we used a set of experimental and computational tools to evaluate the intrinsic disorder status of CALR and the role of calcium binding on structural properties and conformational stability of the full-length CALR and its isolated P- and C-domains.     

The EGF receptor: a nexus for trafficking and signaling

Ligand binding to the EGF receptor initiates both the activation of mitogenic signal transduction pathways plus trafficking events that relocalize the receptor on the cell surface and within intracellular compartments. The trafficking compartments include caveolae, clathrin-coated pits, and various endosome populations prior to receptor degradation in lysosomes. Evidence is presented that distinct signaling pathways are initiated from these different compartments. These include the Ras/MAP kinase cascade and the PLC-dependent hydrolysis of PI-4,5 P(2). Multiple tyrosine kinase substrates that facilitate EGF receptor trafficking between these various compartments, as well as the participation of phosphoinositides and Ras-like G proteins in the trafficking pathway are also described.     

Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites.

Transferrins are two-domain proteins with a very strong site for iron binding located in each domain. Using ultrasensitive titration calorimetry, the binding of ferric ion (chelated with a 2-fold molar excess of nitrilotriacetate) to the two sites of ovotransferrin was studied in detail as well as the binding to the single site in the N- and C-terminal half-molecules. In the presence of excess bicarbonate ion, the binding occurs in two kinetic steps. The fast process of contact binding is instantaneous with respect to instrument response time, is strongly exothermic for the N site and less so for the C site, and corresponds to binding of the chelated ferric ion. The slower process of bicarbonate insertion with concomitant release of nitrilotriacetate occurs on a time scale of 2-20 min over the temperature range 7-37 degrees C and is endothermic for the N site and exothermic for the C site, with rates being significantly slower for insertion at the C site. The delta H of binding is strongly temperature-dependent for both sites, arising from a large negative delta Cp of binding which probably indicates removal of hydrophobic groups from contact with water. When bicarbonate ion is absent, only the fast process of contact binding is seen. Each site within a half-molecule is qualitatively similar to the same site in intact ovotransferrin, although quantitative differences were detected. It was shown that contact binding to ovotransferrin occurs reversibly with free exchange of Fe+3 between N and C sites, while the attachment to either site becomes essentially irreversible after bicarbonate insertion. The strong preference for the first ferric ion to bind to the N site is shown to be due to its larger contact binding constant and the faster rate of bicarbonate insertion, relative to the C site, and is not due to stronger thermodynamic binding after bicarbonate insertion. True equilibrium is achieved only over much longer periods of time. In another series of experiments, direct binding studies were carried out between the two half-molecules under different states of ligation with Fe+3 in the presence of bicarbonate. The results indicate that the two binding sites in ovotransferrin, separated by ca. 40 A, are not independent of one another but communicate as a result of ligand-dependent changes in the heats and free energies of domain-domain interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of heparan sulfate from human primary osteoblasts

Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts—soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non‐identical by a number of parameters, and that these differences have significant ramifications for their ligand‐binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS‐dependent factors between the ECM and the cell surface. J. Cell. Biochem. 108: 1132–1142, 2009. © 2009 Wiley‐Liss, Inc.     

The membrane proteome of stroma thylakoids from Arabidopsis thaliana studied by successive in‐solution and in‐gel digestion

From individual localization and large‐scale proteomic studies, we know that stroma‐exposed thylakoid membranes harbor part of the machinery performing the light‐dependent photosynthetic reactions. The minor components of the stroma thylakoid proteome, regulating and maintaining the photosynthetic machinery, are in the process of being unraveled. In this study, we developed in‐solution and in‐gel proteolytic digestion methods, and used them to identify minor membrane proteins, e.g. transporters, in stroma thylakoids prepared from Arabidopsis thaliana (L.) Heynh Columbia‐0 leaves. In‐solution digestion with chymotrypsin yielded the largest number of peptides, but in combination with methanol extraction resulted in identification of the largest number of membrane proteins. Although less efficient in extracting peptides, in‐gel digestion with trypsin and chymotrypsin led to identification of additional proteins. We identified a total of 58 proteins including 44 membrane proteins. Almost half are known thylakoid proteins with roles in photosynthetic light reactions, proteolysis and import. The other half, including many transporters, are not known as chloroplast proteins, because they have been either curated (manually assigned) to other cellular compartments or not curated at all at the plastid protein databases. Transporters include ATP‐binding cassette (ABC) proteins, transporters for K⁺ and other cations. Other proteins either have a role in processes probably linked to photosynthesis, namely translation, metabolism, stress and signaling or are contaminants. Our results indicate that all these proteins are present in stroma thylakoids; however, individual studies are required to validate their location and putative roles. This study also provides strategies complementary to traditional methods for identification of membrane proteins from other cellular compartments.     

Study of follitropin receptors in testis using a homologous system. Binding of porcine follitropin to plasma membranes from immature porcine testis and correlation with adenylate cyclase stimulation.

The properties of follitropin receptors in immature porcine testis were determined using highly purified porcine follitropin. 1. The characteristics of follitropin binding to a subcellular fraction rich in plasma membranes were studied using a 125I-labelled follitropin with high specific activity (75-100 Ci/g) and high binding activity. The binding is dependent on time, temperature and pH. It is specific to follitropin as demonstrated by the very low binding activity of the follitropin alpha and beta subunits and of the other glycoprotein hormones. Scatchard analysis of binding data indicated an equilibrium association constant of 2 x 10(10) M-1 and a concentration of high affinity binding sites of 500 fmol/mg membrane proteins. 2. A sensitive radio-ligand receptor assay was developed. Fifty percent inhibition of binding was obtained with as little as 2 ng of porcine follitropin. Ovine and bovine follitropins and pregnant mare serum gonadotropin gave binding inhibition curves parallel to that given by porcine follitropin. With equine and human follitropin, significantly different slopes were recorded. 3. Kinetics of dissociation of labelled follitropin from its testis receptors showed the presence of at least two compartments with fast and slow dissociation rate constants. The ratio between the sizes of the slow and fast compartments appeared dependent upon preincubation time. 4. A temporal correlation was observed between binding of follitropin to testis receptors and activation of membrane bound adenylate cyclase.     

New Organelles by Gene Duplication in a Biophysical Model of Eukaryote Endomembrane Evolution

Extant eukaryotic cells have a dynamic traffic network that consists of diverse membrane-bound organelles exchanging matter via vesicles. This endomembrane system arose and diversified during a period characterized by massive expansions of gene families involved in trafficking after the acquisition of a mitochondrial endosymbiont by a prokaryotic host cell >1.8 billion years ago. Here we investigate the mechanistic link between gene duplication and the emergence of new nonendosymbiotic organelles, using a minimal biophysical model of traffic. Our model incorporates membrane-bound compartments, coat proteins and adaptors that drive vesicles to bud and segregate cargo from source compartments, and SNARE proteins and associated factors that cause vesicles to fuse into specific destination compartments. In simulations, arbitrary numbers of compartments with heterogeneous initial compositions segregate into a few compositionally distinct subsets that we term organelles. The global structure of the traffic system (i.e., the number, composition, and connectivity of organelles) is determined completely by local molecular interactions. On evolutionary timescales, duplication of the budding and fusion machinery followed by loss of cross-interactions leads to the emergence of new organelles, with increased molecular specificity being necessary to maintain larger organellar repertoires. These results clarify potential modes of early eukaryotic evolution as well as more recent eukaryotic diversification.     

Predicting the behaviour and selectivity of fluorescent probes for lysosomes and related structures by means of structure-activity models

Summary Cultured rat fibroblasts were exposed to 50 fluorescent probes of varied physicochemical characteristics. Probe concentrations, fluorochrome excitation wavelength and period of illumination, and cell-probe contact time were varied. Structure-activity relationships defining a number of classes of fluorescent probes for lysosomes and related processes and compartments were demonstrated. Numerical specifications are now available for several familiar classes of probes: (a) acidotropic weak bases, used as markers for low pH compartments; (b) markers of adsorptive pinocytosis, involving non-specific protein binding; (c) markers for fluid phase pinocytosis; and (d) viability stains involving intralysosomal enzymic activity. Two novel classes of probes have also been specified numerically: (a) acid-precipitated weak acids, as markers for low pH compartments; and (b) lipid-binding markers of adsorptive pinocytosis. Overall, these structure-activity models provide a tool for predicting whether or not compounds enter cells; and whether they accumulate in lysosomes and related compartments. Pathways of entry are also predicted. This tool should permit design and selection of improved probes, and provide a better understanding of existing reagents. Moreover these models are expected to be applicable to interactions between any non-polymeric xenobiotic with lysosomes and related compartments.     

Getting out: protein traffic in prokaryotes

Protein secretion systems in prokaryotes are increasingly shifting from being considered as experimental models for 'more complex' processes (i.e. eukaryotes) to being a major source of key biological questions in their own right. The pathways by which proteins move between compartments or insert into membranes in prokaryotic cells are certainly less numerous than in eukaryotes (though not dramatically so). However, the quality and complexity of bacterial protein targeting systems indicate that virtually all mechanistic problems associated with protein traffic were solved very efficiently well before eukaryotes appeared on the Earth crust. Indeed, recent studies have both increased the number of known prokaryotic protein traffic systems and indicated new layers of complexity for those that were already well characterized. This report describes some recent developments in bacterial protein traffic that were presented at two meetings in the autumn of 2003.