Antifreeze proteins modify the freezing process in planta

During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 degrees C to 1.2 degrees C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.     

Antifreeze proteins

Antifreeze proteins comprise a structurally diverse class of proteins that inhibit the growth of ice. Recently, new AFP types have been discovered; more active AFPs have been isolated; antecedents have been recognized supporting the notion of recent, multiple origins; and detailed structures have emerged leading to models for their adsorption to ice     

Formation of muscle proteins during freezing

The effect of different conditions on the formation and properties of cryogels prepared by the freezing-thawing procedure from suspensions and solutions of the carp (Cyprinus carpio) myofibrillar proteins was studied. The freezing of water solutions and suspensions of the native myofibrillar proteins resulted in the formation of the structures mainly stabilized by non-covalent bonds. When muscle proteins were denatured prior to the freezing they formed the structures stabilized by both non-covalent and covalent disulfide bonds.     

Small heat shock proteins differentially affect Abeta aggregation and toxicity

beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and has been implicated in neurotoxicity associated with the disease. Abeta aggregates readily in vitro and in vivo, and its toxicity has been linked to its aggregation state. Prevention of Abeta aggregation has been investigated as a means to prevent Abeta toxicity associated with AD. Recently we found that Hsp20 from Babesia bovis prevented both Abeta aggregation and toxicity [S. Lee, K. Carson, A. Rice-Ficht, T. Good, Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity, Protein Sci. 14 (2005) 593-601.]. In this work, we examined the mechanism of Hsp20 interaction with Abeta1-40 and compared its activity to that of other small heat shock proteins, carrot Hsp17.7 and human Hsp27. While all three small heat shock proteins were able to prevent Abeta aggregation, only Hsp20 was able to attenuate Abeta toxicity in cultured SH-SY5Y cells. Understanding the mechanism of the Hsp20-Abeta interaction may provide insights into the design of the next generation of Abeta aggregation and toxicity inhibitors.     

Antifreeze proteins in higher plants

Overwintering plants produce antifreeze proteins (AFPs) having the ability to adsorb onto the surface of ice crystals and modify their growth. Recently, several AFPs have been isolated and characterized and five full-length AFP cDNAs have been cloned and characterized in higher plants. The derived amino acid sequences have shown low homology for identical residues. Theoretical and experimental models for structure of Lolium perenne AFP have been proposed. In addition, it was found that the hormone ethylene is involved in regulating antifreeze activity in response to cold. In this review, it is seen that the physiological and biochemical roles of AFPs may be important to protect the plant tissues from mechanical stress caused by ice formation.     

Antifreeze proteins in winter rye

Six antifreeze proteins, which have the unique ability to adsorb onto the surface of ice and inhibit its growth, have been isolated from the apoplast of winter rye leaves where ice forms at subzero temperatures. The rye antifreeze proteins accumulate during cold acclimation and are similar to plant pathogenesis-related proteins, including two endoglucanase-like, two chitinase-like and two thaumatin-like proteins. Immunolocalization of the glucanase-like antifreeze proteins showed that they accumulate in mesophyll cell walls facing intercellular spaces, in pectinaceous regions between adjoining mestome sheath cells, in the secondary cell walls of xylem vessels and in epidermal cell walls. Because the rye antifreeze proteins are located in areas where they could be in contact with ice, they may function as a barrier to the propagation of ice or to inhibit the recrystallization of ice. Antifreeze proteins similar to pathogenesis-related proteins were also found to accumulate in closely-related plants within the Triticum group but not in freezing-tolerant dicotyledonous plants. In winter wheat, the accumulation of antifreeze proteins and the development of freezing tolerance are regulated by chromosome 5. Rye antifreeze proteins may have evolved from pathogenesis-related proteins, but they retain their catalytic activities and may play a dual role in increasing both freezing and disease resistance in overwintering plants.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between − 3 and − 10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below − 4 °C and cell survival exhibits an optimum at a nucleation temperature of − 6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at − 3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at − 10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 degrees C/min while the ice nucleation temperature was varied between -3 and -10 degrees C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below -4 degrees C and cell survival exhibits an optimum at a nucleation temperature of -6 degrees C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at -3 degrees C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at -10 degrees C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in alpha-helical structures and a concomitant increase in beta-sheet structures starting at an onset temperature of approximately 48 degrees C.     

Membrane-associated proteins affect the formation of filipin-cholesterol complexes in viral membranes

The presence and distribution of cholesterol in biological membranes can be visualized by complex formation with the polyene antibiotic filipin after or during fixation with glutaraldehyde. In the envelopes of budding and immature retroviruses no filipin-cholesterol complexes are formed, but in the plasma membrane of host cells and in the envelopes of mature viruses filipin-cholesterol complexes are easily detected. However, after treatment of glutaraldehyde-fixed cells with pepsin, the presence of cholesterol in the envelopes of budding and immature viral particles could also be demonstrated. This indicates that in these structures the reaction of cholesterol with filipin is inhibited by proteins associated with the cholesterol-containing membrane. Treatment of fixed cells with trypsin, and of unfixed cells with cytochalasin B (CB) had no effect on detectability of cholesterol in these structures. On no occasion were cholesterol-filipin complexes formed in coated pits. The present findings call for caution when interpretating data on absence of filipincholesterol complexes in those membrane domains that are characterized by the presence of closely associated proteins.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.     

Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.     

Antifreeze proteins: an unusual receptor-ligand interaction

Antifreeze proteins (AFPs) help organisms to survive below 0 degrees C by inhibiting ice growth. Although AFPs are structurally diverse, they typically present a large proportion of their surface area for binding to ice. Whereas earlier proposed binding mechanisms relied almost entirely on a hydrogen bond match between the AFP and ice, it now seems probable that van der Waals and hydrophobic interactions make a significant contribution to the enthalpy of adsorption. These interactions require intimate surface-surface complementarity between the receptor (AFP) and its ligand (ice).     

Antifreeze proteins in Alaskan insects and spiders

Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hysteresis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (-40 to -70 degrees C).     

Antifreeze proteins bind independently to ice.

It has been suggested that cooperative interactions between antifreeze proteins (AFPs) on the ice surfaces are required for complete inhibition of ice crystal growth. To test this hypothesis, a 7-kDa type III AFP was linked through its N-terminus to thioredoxin (12 kDa) or maltose-binding protein (42 kDa). The resultant 20-kDa and 50-kDa fusion proteins were larger in diameter than free AFP and thus precluded any extensive AFP-AFP contacts on the ice surface. Both fusion proteins were at least as active as free AFP at virtually all concentrations tested. By these criteria, AFPs function independently of each other and do not require specific intermolecular interactions to bind tightly to ice.     

Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal β-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.     

Monensin and brefeldin A differentially affect the phosphorylation and sulfation of secretory proteins.

Chromogranin B and secretogranin II, two members of the granin family, are known to be post-translationally modified by the addition of O-linked carbohydrates to serine and/or threonine, phosphate to serine and threonine, and sulfate to carbohydrate and tyrosine residues. In the present study, chromogranin B and secretogranin II were used as model proteins to investigate in which subcompartment of the Golgi complex secretory proteins become phosphorylated. Monensin, a drug known to block the transport from the medial to the trans cisternae of the Golgi stack, inhibited the phosphorylation of the granins, indicating that this modification occurred distal to the medial Golgi. Monensin also blocked the addition of galactose to O-linked carbohydrates and the sulfation of the granins, confirming previous data that these modifications take place in the trans Golgi. To distinguish, within the trans Golgi, between the trans cisternae of the Golgi stack and the trans Golgi network, we made use of the previous observation that brefeldin A results in the redistribution to the endoplasmic reticulum of membrane-bound enzymes of the trans cisternae of the Golgi stack, but not of the trans Golgi network. Brefeldin A treatment abolished granin sulfation but resulted in the accumulation of phosphorylated and galactosylated granins. Differential effects of brefeldin A on membranes of the Golgi stack versus the trans Golgi network were also observed by immunofluorescence analysis of marker proteins specific for either compartment. Our results suggest that the phosphorylation of secretory proteins, like their galactosylation, largely occurs in the trans cisternae of the Golgi stack, whereas the sulfation of secretory proteins on both carbohydrate and tyrosine residues takes place selectively in the trans Golgi network.     

Identification of proteins differentially expressed during chondrogenesis of mesenchymal cells

We performed comparative proteome analysis of mesenchymal cells and chondrocytes to identify proteins differentially expressed during chondrogenesis. Nine such proteins were identified. Type II collagen, matrilin-1, carbonic anhydrase-II (CA-II), 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2, and aldo-keto reductase were increased during chondrogenesis, whereas cellular retinoic acid binding protein-I (CRABP-I), CRABP-II, cytoplasmic type 5 actin, and fatty acid binding protein were decreased or almost disappeared. Expression of type II collagen, matrilin-1, PAPS synthetase-2, and CA-II was regulated by extracellular signal-regulated protein kinase, protein kinase C, and p38 kinase, signaling molecules known to regulate chondrogenesis.     

Cryoprotectin: a plant lipid-transfer protein homologue that stabilizes membranes during freezing

Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non-freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold-regulated proteins is not yet known. We summarize our knowledge of those cold-regulated proteins that are able to stabilize membranes during a freeze-thaw cycle. Special emphasis is placed on cryoprotectin, a lipid-transfer protein homologue that was isolated from cold-acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze-thaw damage.     

Rb family proteins differentially regulate distinct cell lineages during epithelial development

pRb, p107 and p130 are important regulators of cell cycle and have extensive overlapping functions; however, only Rb has been shown to be a bone fide tumor suppressor. Defining the overlapping versus distinct pocket protein functions is therefore an important step to understanding the unique role of Rb. Using lung as a model, the present studies demonstrate that pocket proteins are important not only in regulating cell cycle and survival but also in cell lineage specification. An inducible lung-specific Rb knockout strategy was used to demonstrate that Rb is specifically required for restricting neuroendocrine cell fate despite functional compensation for Rb deficiency in other cell types. Ablation of total Rb family function resulted in opposing effects in specification along distinct cell lineages, providing evidence that pocket proteins inhibit neuroendocrine cell fate while being required for differentiation in other cell types. These findings identify a novel role for pocket proteins in cell fate determination, and establish a unique cell lineage-specific function for Rb that explains, at least in part, why Rb and p16 are inactivated in phenotypically distinct carcinomas.     

Identification of differentially expressed proteins during larval molting of Helicoverpa armigera

Insect molting involves many molecular processes, such as protein degradation and protein synthesis in the epidermis. Various proteins have been implicated in these processes. The differentially expressed proteins during larval molting of Helicoverpa armigera were investigated using two-dimensional electrophoresis (2-D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALTI-TOF-MS). Four larval tissues sampled during molting and feeding were examined. Seventy-seven differentially expressed proteins were identified in these tissues, including 20 proteins from the fifth-molting epidermis (fifth instar molting to sixth instar), 36 proteins from the fifth-molting hemolymph, and 21 from the fifth-molting fat bodies. No obviously different spots were identified from the fifth-molting midgut under these experimental conditions. After application of MALTI-TOF-MS and similarity analysis comparing results to a Drosophila protein database, 30 proteins were identified: 10 proteins from the fifth-molting epidermis, 11 proteins from the hemolymph, and 9 proteins from fat bodies. These proteins were separated into 5 groups according to their probable functions, such as enzymes, regulators, protein hydrolases, receptors, and proteins with unknown functions. These differentially expressed proteins were proposed to be involved in the Helicoverpa molting cascade.