Water penetration and binding to ferric myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.     

Membrane proteins: amino acid sequence and membrane penetration

A computer study shows that the membrane-penetrating portion of the erythrocyte surface MN-glycoprotein (Winzler, 1969; Marchesi et al., 1972) is distinguishable by informal cluster analysis from other segments of globular proteins when sequence length is plotted against hydrophobicity This analysis further suggests the possibility that other membrane-penetrating segments of proteins can be identified in the same way.     

Intrinsically disordered proteins may escape unwanted interactions via functional misfolding

Intrinsically disordered proteins are highly abundant in nature and play a number of crucial roles in the living cells. They are commonly involved in a wide range of intermolecular interactions, and some of them possess remarkable binding promiscuity, being able to interact specifically with structurally unrelated partners. Although they do not have well-folded structure, some IDPs are known to fold at binding to their specific partners. IDPs are highly pliable and one IDP can form an array of unrelated structures being bound to different partners. It is believed that many IDPs, being mostly disordered, have transient elements of the preformed secondary structure which are highly interaction prone and is used by IDPs for binding to specific partners. The overall disordered nature of IDPs, their high conformational dynamics and flexibility, the presence of sticky preformed binding elements, and their ability to morph into differently-shaped bound configurations raised a very important question about the mechanisms preventing IDPs from unwanted interactions with non-native partners. In this review, a concept of functional misfolding is introduced. Accumulated to date data on the conformational behavior and fine structure of several IDPs suggest that the preformed binding elements might be involved in a set of non-native intramolecular interactions. In other words, there is a chance that a polypeptide chain misfolds to sequester the preformed elements inside the non-interactive or less-interactive cage, therefore preventing these elements from the unnecessary and unwanted interactions with non-native binding partners.     

Adenovirus capsid proteins interact with HSP70 proteins after penetration in human or rodent cells

Soon after penetration of adenovirus serotype 2 in BHK-21 and HeLa cells, HSP70 and HSC70 proteins become associated with the viral capsid. By analysis with a polyclonal antibody derived from a fusion protein containing the C-terminal domain, 290 amino acids of HSP70, and using both immunological methods and infected cells fractionation we observed that a significant amount of HSP70 proteins moved to the nucleus and colocalized with the adenovirus particles. HSP70 proteins of infected cells were isolated as a complex cross-linked with intracytoplasmic adenovirus type 2. By coprecipitation, using a polyclonal-specific antiserum derived from the fusion protein, or two different monoclonal-specific antisera, we showed that HSP70 and HSC70 proteins were associated with hexon, the major adenovirus capsid protein.     

Water penetration in the low and high pressure native states of ubiquitin

Theoretical studies on the solvation of methane molecules in water have shown that the effect of increased pressure is to stabilize solvent separated contacts relative to direct contacts. This suggests that high pressure stabilizes waters that have penetrated into a protein's core, indicating a mechanism for the high pressure denaturation of proteins. We test this theory on a folded protein by studying the penetration of water into the native state of ubiquitin at low and high pressures, using molecular dynamics. An ensemble of conformations sampled in the folded state of ubiquitin has been determined by NMR at two pressures below the protein's denaturation pressure, 30 atm and 3000 atm. We find that 1-5 more waters penetrate the high pressure conformations than the low pressure conformations. Low volume configurations of the system are favored at high pressures, but different components of the system may experience increases or decreases in their specific volumes. We find that penetrating waters have a higher volume per water than bulk waters, but that the volume per protein residue may be lowered by solvation. Furthermore, we find that penetration of the protein by water at high pressures is driven by the difference in the pressure dependence of the probability of cavity opening in the protein and pressure dependence of the probability of cavity opening in the bulk solvent. The volume changes associated with cavity opening and closing indicate that each penetrating water reduces the volume of the system by about 12 mL/mol. The experimental volume change going from the low pressure to the high pressure native state of ubiquitin is 24 mL/mol. Our results indicate that this volume change can be explained by penetration of the protein by two water molecules.     

Antiapoptotic herpesvirus Bcl-2 homologs escape caspase-mediated conversion to proapoptotic proteins

The antiapoptotic Bcl-2 and Bcl-x(L) proteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (E. H.-Y. Cheng, D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno, and J. M. Hardwick, Science 278:1966-1968, 1997; R. J. Clem, E. H.-Y. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 95:554-559, 1998). Gamma herpesviruses also encode homologs of the Bcl-2 family. All tested herpesvirus Bcl-2 homologs possess antiapoptotic activity, including the more distantly related homologs encoded by murine gammaherpesvirus 68 (gammaHV68) and bovine herpesvirus 4 (BHV4), as described here. To determine if viral Bcl-2 proteins can be converted into death factors, similar to their cellular counterparts, five herpesvirus Bcl-2 homologs from five different viruses were tested for their susceptibility to caspases. Only the viral Bcl-2 protein encoded by gammaHV68 was susceptible to caspase digestion. However, unlike the caspase cleavage products of cellular Bcl-2, Bcl-x(L), and Bid, which are potent inducers of apoptosis, the cleavage product of gammaHV68 Bcl-2 lacked proapoptotic activity. KSBcl-2, encoded by the Kaposi's sarcoma-associated herpesvirus, was the only viral Bcl-2 homolog that was capable of killing cells when expressed as an N-terminal truncation. However, because KSBcl-2 was not cleavable by caspases, the latent proapoptotic activity of KSBcl-2 apparently cannot be released. The Bcl-2 homologs encoded by herpesvirus saimiri, Epstein-Barr virus, and BHV4 were not cleaved by apoptotic cell extracts and did not possess latent proapoptotic activities. Thus, herpesvirus Bcl-2 homologs escape negative regulation by retaining their antiapoptotic activities and/or failing to be converted into proapoptotic proteins by caspases during programmed cell death.     

Geometric differences in the ribosome exit tunnel impact the escape of small nascent proteins

The exit tunnel is the subcompartment of the ribosome that contains the nascent polypeptide chain and, as such, is involved in various vital functions, including regulation of translation and protein folding. As the geometry of the tunnel shows important differences across species, we focus on key geometrical features of eukaryote and prokaryote tunnels. We used a simple coarse-grained molecular dynamics model to study the role of the tunnel geometry in the post-translational escape of short proteins (short open reading frames [sORFs]) with lengths ranging from 6 to 56 amino acids. We found that the probability of escape for prokaryotes is one for all but the 12-mer chains. Moreover, proteins of this length have an extremely low escape probability in eukaryotes. A detailed examination of the associated single trajectories and energy profiles showed that these variations can be explained by the interplay between the protein configurational space and the confinement effects introduced by the constriction sites of the ribosome exit tunnel. For certain lengths, either one or both of the constriction sites can lead to the trapping of the protein in the “pocket” regions preceding these sites. As the distribution of existing sORFs indicates some bias in length that is consistent with our findings, we finally suggest that the constraints imposed by the tunnel geometry have impacted the evolution of sORFs.     

Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis

The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.     

Water channel proteins in the gastrointestinal tract

Water transport through the human digestive system is physiologically crucial for maintaining body water homeostasis and ensure digestive and absorptive functions. Within the gastrointestinal tract, water recirculates, being secreted with the digestive juices and then almost entirely absorbed by the small and large intestine. The importance of aquaporins (AQPs), transmembrane water channel proteins, in the rapid passage of water across plasma membranes in the gastrointestinal tract appears immediately evident. Several AQP isoforms are found in gastrointestinal epithelia, with AQP1, 3, 7, 10 and 11 being the most abundantly expressed in the whole gut. On the other hand, AQP4 and 8 are located selectively in the stomach and colon, respectively. Here we review AQP expression and localization at the tissue, cellular and subcellular level in gastrointestinal epithelia, and their modification in various gut diseases.     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

Live Salmonella modulate expression of Rab proteins to persist in a specialized compartment and escape transport to lysosomes

We investigated the intracellular route of Salmonella in macrophages to determine a plausible mechanism for their survival in phagocytes. Western blot analysis of isolated phagosomes using specific antibodies revealed that by 5 min after internalization dead Salmonella-containing phagosomes acquire transferrin receptors (a marker for early endosomes), whereas by 30 min the dead bacteria are found in vesicles carrying the late endosomal markers cation-dependent mannose 6-phosphate receptors, Rab7 and Rab9. In contrast, live Salmonella-containing phagosomes (LSP) retain a significant amount of Rab5 and transferrin receptor until 30 min, selectively deplete Rab7 and Rab9, and never acquire mannose 6-phosphate receptors even 90 min after internalization. Retention of Rab5 and Rab18 and selective depletion of Rab7 and Rab9 presumably enable the LSP to avoid transport to lysosomes through late endosomes. The presence of immature cathepsin D (48 kDa) and selective depletion of the vacuolar ATPase in LSP presumably contributes to the less acidic pH of LSP. In contrast, proteolytically processed cathepsin D (M(r) 17,000) was detected by 30 min on the dead Salmonella-containing phagosomes. Morphological analysis also revealed that after uptake by macrophages, the dead Salmonella are transported to lysosomes, whereas the live bacteria persist in compartments that avoid fusion with lysosomes, indicating that live Salmonella bypass the normal endocytic route targeted to lysosomes and mature in a specialized compartment.     

Herpes simplex virus type 1 penetration initiates mobilization of cell surface proteins

Changes in membrane structure resulting from herpes simplex virus 1 (HSV-1) penetration were detected using fluorescence photobleaching recovery methods. The effect could be blocked by inhibitors of viral and cellular processes involved in virus penetration. A rapid mode of HSV-1 strain KOS penetration into VERO cells at 37 degrees C normally occurs after a 5 min lag period and is 90-95% complete within 20-30 min. Rates of cell surface protein diffusion increase 2-3-fold after 5 min and return to normal after 25-30 min, this return correlating temporally with the penetration of the virus. At pH 6.3 the lag period preceeding penetration of HSV is increased to 20 min and penetration proceeds much more slowly than at pH 7.4. Inhibition of virus penetration with cytochalasin B or with the antiherpes drug tromantadine also prevents the HSV-1-induced increase in cell surface protein mobility. Colchicine, which does not block HSV-1 penetration, prevents the recovery of the membrane following virus penetration. Therefore, the changes in membrane structure characterized by increased cell surface protein mobility seem to be caused by virus penetration. Cytoskeletal function and integrity are required for the initiation of, and cell recovery from, virus penetration. A pH-sensitive activity, likely to be a virion fusion glycoprotein, is also required.     

Water mobility, denaturation and the glass transition in proteins

A quantitative mechanism is presented that links protein denaturation and the protein-water glass transition through an energy criterion for the onset of mobility of strong protein-water bonds. Differences in the zero point vibrational energy in the ordered and disordered bonded states allow direct prediction of the two transition temperatures. While the onset of water mobility induces the same change in heat capacity for both transitions, the order-disorder transition of denaturation also predicts the observed excess enthalpy gain. The kinetics of the water and protein components through the glass transition are predicted and compared with dielectric spectroscopy observations. The energetic approach provides a consistent mechanism for processes such as refolding and aggregation of proteins involved in protein maintenance and adaptability, as the conformational constraints of strong water-amide bonds are lost with increased molecular mobility. Moreover, we suggest that the ordered state of peptide-water bonds is induced at the point of protein synthesis and could play a key role in the function of proteins through the enhancement of electronic activity by ferroelectric domains in the protein hydration shell, which is lost upon denaturation.     

X-chromosome inactivation and escape

X-chromosome inactivation, which was discovered by Mary Lyon in 1961 results in random silencing of one X chromosome in female mammals. This review is dedicated to Mary Lyon, who passed away last year. She predicted many of the features of X inactivation, for e.g., the existence of an X inactivation center, the role of L1 elements in spreading of silencing and the existence of genes that escape X inactivation. Starting from her published work here we summarize advances in the field.