A-kinase anchoring proteins: scaffolding proteins in the heart

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called "A-kinase anchoring proteins" (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.     

Characterization of abnormal proteins in the soluble lens proteins of CatFraser mice

The murine mutation CatFraser causes a cataract in both the mutant heterozygote (C/+) and mutant homozygote (C/C). Our previous electrophoretic studies detected, in the lenses of both mutant genotypes, several proteins which did not appear to be present in normal lenses. Here we show, using more sensitive methods, that traces of these proteins previously characterized as abnormal are in fact present in normal lenses. Furthermore, these proteins have a primary structure very similar to that of the alpha-crystallins. We conclude that the mutation accelerates the degradation of alpha-crystallin, which in the normal lens proceeds at a very slow rate.     

Calcium-sensitivity of brain microtubule proteins in the presence of S-100 proteins

In the presence of the usual 0.1 M Mes buffer, pH 6.7, mM free Ca2+ levels are required for half-maximal decrease in the rate and extent of brain microtubule protein (MTP) assembly in the absence of ox brain S-100, while microM free Ca2+ levels are sufficient in the presence of S-100. At the same pH 6.7, but in the presence of 0.12 M KCl, as low as 1.5 microM free Ca2+ is sufficient for S-100 to produce half-maximal reduction in the rate of assembly, while as high as 0.5 mM free Ca2+ is required in the absence of S-100. Similar results are obtained with rat brain S-100 (S-100b), indicating that single S-100 iso forms are equipotent in affecting the MTP assembly. At pH 7.5, MTPs are remarkably resistant to Ca2+ in the absence of S-100. In the presence of S-100, not only is the free Ca2+ concentration required for complete inhibition of assembly at least one order of magnitude smaller than that required in the absence of S-100, but significant S-100-dependent inhibition of assembly occurs in the absence of Ca2+. Under the two conditions where S-100 is particularly effective in inhibiting the assembly, i.e. at pH 6.7 in the presence of KCl and at pH 7.5, S-100 increases the disassembly rate even in the presence of microM Ca2+ levels. Our results suggest that the free Ca2+ concentration regulates the way S-100 disassembles microtubules (MTs): at microM Ca2+ levels, S-100 sequesters tubulin with concomitant increase in the disassembly rate; at mM Ca2+ levels, the S-100-Ca2+ complex probably interacts with MTs producing endwise disassembly.     

Immunolocalization of stress proteins and extracellular matrix proteins in the rat tibia

Stress proteins (heat shock proteins [hsps]) serve a number of protective functions, including protection from apoptosis and acting as chaperones during protein biosynthesis. For example, hsp 27 has been defined as a chaperone for the G3 domain of aggrecan, while hsp 47 is the chaperone for type I collagen. Separate cytoprotective roles for hsp 27 and hsp 70 have been demonstrated. The aim of this study was to define the expression of hsps in osteoblastic and chondrocytic cells of the growing rat long bone in relationship to the immunohistochemical localization of aggrecan, type I collagen and the presence of fragmented DNA that defines apoptotic events. Tibiae were harvested from Fisher 344 rats (n=6) and fixed in 10% buffered formalin. Samples were decalcified in 10% EDTA, bisected, and processed for histologic examination. Sections (5 mm) were immunohistochemically stained using a streptavidin-biotin detection method. Co-localization of hsps with apoptosis was achieved using the TUNEL procedure. In the rat tibia growth plate, aggrecan was generally distributed throughout cartilage and chondrocytes. However, hsp 27 expression was observed only in the lower hypertrophic chondrocytes. hsp27 was present in osteoblasts lining newly formed bone. hsp 47 staining was also prominent within these osteoblasts where collagen type I immunolocalization occurred. The inducible form of hsp 70 was localized to the osteoblastic cells lining new bone in the primary spongiosa. In cartilage, DNA fragmentation was restricted to the hypertrophic, hsp27-positive, chondrocytes. In contrast, DNA fragmentation was not co-localized with hsp27-positive osteoblastic cells of the primary spongiosa, although occasional apoptotic cells were identified. These results indicate that apoptosis is a mechanism by which hypertrophic chondrocytes are eliminated from cartilage prior to calcification, but that other mechanisms are also likely to be involved. They also suggest that hsps have cytoprotective and biosynthetic functions within osteoblasts and chondrocytes, but apoptotic signals may override these effects in some instances, resulting in apoptosis.     

Ribosomal proteins in the spotlight

The assignment of specific ribosomal functions to individual ribosomal proteins is difficult due to the enormous cooperativity of the ribosome; however, important roles for distinct ribosomal proteins are becoming evident. Although rRNA has a major role in certain aspects of ribosomal function, such as decoding and peptidyl-transferase activity, ribosomal proteins are nevertheless essential for the assembly and optimal functioning of the ribosome. This is particularly true in the context of interactions at the entrance pore for mRNA, for the translation-factor binding site and at the tunnel exit, where both chaperones and complexes associated with protein transport through membranes bind.     

Exploring the role of cation-pi interactions in glycoproteins lipid-binding proteins and RNA-binding proteins

We have analyzed and compared the influence of cation-pi interactions in glycoproteins (GPs), lipid-binding proteins (LBPs) and RNA-binding proteins (RBPs) in this study. We observed that all the proteins included in the study had profound cation-pi interactions. There is an average of one energetically significant cation-pi interaction for every 71 residues in GPs, for every 58 residues in LBPs and for every 64 residues in RBPs. Long-range contacts are predominant in all the three types of proteins studied. The pair-wise cation-pi interaction energy between the positively charged and aromatic residues shows that Arg-Trp pair energy was the strongest among all six possible pairs in all the three types of proteins studied. There were considerable differences in the preference of cation-pi interacting residues to different secondary structure elements and ASA and these might contribute to differences in biochemical functions of GPs, LBPs and RBPs. It was interesting to note that all the five residues involved in cation-pi interactions were found to have stabilization centers in GPs, LBPs and RBPs. Majority of the cation-pi interacting residues investigated in the present study had a conservation score of 6, the cutoff value used to identify the stabilizing residues. A small percentage of cation-pi interacting residues were also present as stabilizing residues. The cation-pi interaction-forming residues play an important role in the structural stability of in GPs, LBPs and RBPs. The results obtained in this study will be helpful in further understanding the stability, specificity and differences in the biochemical functions of GPs, LBPs and RBPs.     

Using model proteins to quantify the effects of pathogenic mutations in Ig-like proteins

It has proved impossible to purify some proteins implicated in disease in sufficient quantities to allow a biophysical characterization of the effect of pathogenic mutations. To overcome this problem we have analyzed 37 different disease-causing mutations located in the L1 and IL2Rgamma proteins in well characterized related model proteins in which mutations that are identical or equivalent to pathogenic mutations were introduced. We show that data from these models are consistent and that changes in stability observed can be correlated to severity of disease, to correct trafficking within the cell and to in vitro ligand binding studies. Interestingly, we find that any mutations that cause a loss of stability of more than 2 kcal/mol are severely debilitating, even though some model proteins with these mutations can be easily expressed and analyzed. Furthermore we show that the severity of mutation can be predicted by a DeltaDeltaG(evolution) scale, a measure of conservation. Our results demonstrate that model proteins can be used to analyze disease-causing mutations when wild-type proteins are not stable enough to carry mutations for biophysical analysis.     

Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins

Retinal phosducin is known to sequester transducin Gbetagamma, thereby modulating transducin activity. Phosducin is a member of a family of phosducin-like proteins (PhLP) found in eukaryotes. Phylogeny of 33 phosducin-like proteins from metazoa, plants and lower eukaryotes identified three distinct groups named phosducin-I-III. We discovered three phlp genes in Dictyostelium, each encoding a phosducin-like protein of a different group. Disruption of the phlp1 gene strongly impaired G-protein signalling, apparently due to mislocalization of Gbetagamma in phlp1-null cells. GFP-Gbeta and GFP-Ggamma are membrane associated in wild-type cells, but cytosolic in phlp1-null cells. Phlp2 disruption is lethal due to a synchronous collapse of the cells after 16-17 cell divisions. Phlp3 disruptants show no abnormal phenotype. These results establish a role for phosducin-like proteins in facilitating folding, localization or function of proteins, in addition to modulating G-protein signalling.     

NMR of membrane proteins in micelles and bilayers: the FXYD family proteins

Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.     

Characterization of the secretory proteins of rat preputial gland in relation to urinary proteins

The water-soluble proteins of the rat preputial gland secretion were characterized in native and SDS-treated form on polyacrylamide gel electrophoresis. Nine major proteins were present in the secretion. One protein was a glycoprotein of molecular weight greater than 200,000 with beta-glucuronidase activity, and the other eight proteins had a molecular weight of 17,000, but with different charges. Acid phosphatase and arylsulphatase activities were present in the secretion in minor amounts. The isoelectric points of the secretory proteins ranged from 8.5 to 5.3; none of the proteins were lipoproteins, and there were no sex differences. The male and female rat urinary proteins were also characterized electrophoretically. The male rats had two different protein patterns, probably genetically determined. The female rats showed basically one urinary protein pattern, but their urines were frequently mixed with the preputial gland secretory proteins, which most likely played a part in the chemical communication. The mixing could not be correlated to daytime or estrous cycle.