Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].     

Climate change impacts on potential recruitment in an ecosystem engineer

Climate variability and the rapid warming of seas undoubtedly have huge ramifications for biological processes such as reproduction. As such, gametogenesis and spawning were investigated at two sites over 200 km apart on the south coast of Ireland in an ecosystem engineer, the common cockle, Cerastoderma edule. Both sites are classed as Special Areas of Conservation (SACs), but are of different water quality. Cerastoderma edule plays a significant biological role by recycling nutrients and affecting sediment structure, with impacts upon assemblage biomass and functional diversity. It plays a key role in food webs, being a common foodstuff for a number of marine birds including the oystercatcher. Both before and during the study (early 2010–mid 2011), Ireland experienced its two coldest winters for 50 years. As the research demonstrated only slight variation in the spawning period between sites, despite site differences in water and environmental quality, temperature and variable climatic conditions were the dominant factor controlling gametogenesis. The most significant finding was that the spawning period in the cockle extended over a greater number of months compared with previous studies and that gametogenesis commenced over winter rather than in spring. Extremely cold winters may impact on the cockle by accelerating and extending the onset and development of gametogenesis. Whether this impact is positive or negative would depend on the associated events occurring on which the cockle depends, that is, presence of primary producers and spring blooms, which would facilitate conversion of this extended gametogenesis into successful recruitment.     

Cognitive Bias of Interpretation in Type D Personality: Associations with Physiological Indices of Arousal

It is argued that the Type D person experiences increased distress when encountering social situations, which results in altered stress responding. However, little is known about how Type D individuals behave or feel during social interactions. Using a within-subjects design, the present study examined the physiological arousal of Type D and non-Type D individuals when rating how they would deal with a number of hypothetical social situations. Results showed that across all social situations, Type D individuals experienced greater levels of discomfort. In clearly negative and ambiguously neutral situations, this was paired with higher pulse rate. This study shows that the Type D individual not only reports greater discomfort but also experiences changes physiologically that support the role of the cardiovascular system as a potential psychosomatic pathway to disease.     

A Forward Genetic Screen Targeting the Endothelium Reveals a Regulatory Role for the Lipid Kinase Pi4ka in Myelo- and Erythropoiesis

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Aß Facilitates LTD at Schaffer Collateral Synapses Preferentially in the Left Hippocampus

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Cystic Fibrosis: Channel, Catalytic, and Folding Properties of the CFTR Protein

The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large supeifamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bi-directional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.     

Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases

Cystic fibrosistransmembrane conductance regulator (CFTR)Cl channel activitydeclines rapidly when excised from transfected Chinese hamster ovary(CHO) or human airway cells because of membrane-associated phosphataseactivity. In the present study, we found that CFTR channels usuallyremained active in patches excised from baby hamster kidney (BHK) cellsoverexpressing CFTR. Those patches with stable channel activity wereused to investigate the regulation of CFTR by exogenous proteinphosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase toexcised patches reduced CFTR channel activity by >90% but did notabolish it completely. PP2B caused weak deactivation, whereas PP1 hadno detectable effect on open probability(P_o).Interestingly, the time course of deactivation by PP2C was identical tothat of the spontaneous rundown observed in some patches afterexcision. PP2C and PP2A had distinct effects on channel gating;P_o declinedduring exposure to exogenous PP2C (and during spontaneous rundown, whenit was observed) without any change in mean burst duration. Bycontrast, deactivation by exogenous PP2A was associated with a dramaticshortening of burst duration similar to that reported previously inpatches from cardiac cells during deactivation of CFTR by endogenousphosphatases. Rundown of CFTR-mediated current across intact T84epithelial cell monolayers was insensitive to toxic levels of the PP2Ainhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and thatmultiple protein phosphatases may be required for complete deactivationof CFTR channels.

     

The binding of asialo-glycoprotein to isolated Golgi apparatus

Membranes of the Golgi apparatus isolated from rat liver were capable of binding ¹²⁵I-asialo-fetuin in a manner similar to the binding to liver plasma membranes. Although the binding capacity of the Golgi membranes was less than that of plasma membranes, binding was dependent on Ca⁺⁺ ions and inhibited by α-lactalbumin in both cases. Specific activities of galactosyl and sialyl transferases were about 20 times greater in Golgi than in plasma membranes isolated from the same livers. This dramatic reciprocal relationship between enzyme levels and binding capacities of the two membranous fractions argues against either of these enzymes being the actual binding site.