Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.

Ezrin and moesin are components of actin-rich cell surface structures that are thought to function as membrane-cytoskeletal linking proteins. Here we show that a stable complex of ezrin and moesin can be isolated from cultured cells by immunoprecipitation with specific antibodies. The capacity of these two proteins to interact directly was confirmed with a blot-overlay procedure in which biotin-tagged proteins in solution were incubated with immobilized binding partners. In addition to the heterotypic association of ezrin and moesin, homotypic binding of ezrin to ezrin and of moesin to moesin was also demonstrated in vitro. These results suggest mechanisms by which ezrin and moesin might participate in dynamic aspects of cortical cytoskeletal structure.     

The A-type lamins: nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.

Mutations in the lamin A (LMNA) gene are associated with the tissue-specific diseases Emery-Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy (LGMD-1B), dilated cardiomyopathy with conduction system disease (DCM-CD), and Dunnigan's familial partial lipodystrophy (FPLD). Lamins A and C, the products of the LMNA gene, are nuclear intermediate filament proteins and are the major structural components of the lamina network that underlies and supports the nuclear envelope. Nuclear fragility and mislocalization of the nuclear envelope protein emerin are two defects induced by a lack of the A-type lamins. These observations reveal that organization and structural integrity of the nucleus are critical factors in the origins of certain dystrophic and cardiovascular diseases.     

The nuclear pore complex: disease associations and functional correlations.

Nuclear pore complexes (NPCs) are large protein structures spanning the double membrane of the eukaryotic nucleus that serve as sites for translocation of macromolecules between the nucleus and the cytoplasm. The vertebrate NPC has recently been found to comprise approximately 30 distinct proteins, collectively referred to as nucleoporins. Studies over the past several years have demonstrated that individual nucleoporins have unique roles in regulating NPC function and the nucleocytoplasmic transport of proteins and RNAs. The unique functions of individual nucleoporins have been made most clear through their associations with specific human diseases. Here, we highlight the relationships between individual nucleoporins and disease, with particular emphasis given to ALADIN, a nucleoporin linked to a genetically heritable human disease known as triple A syndrome.     

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins.

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.     

Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis

Cytotoxic T lymphocytes (CTL) induce apoptosis by engaging death receptors or by exocytosis of cytolytic granules containing granzyme (Gzm) proteases and perforin. The lamins, which maintain the structural integrity of the nuclear envelope, are cleaved by caspases during caspase-mediated apoptosis. Although death receptor engagement and GzmB activate caspases, CTL also induce apoptosis during caspase blockade. Both GzmA and GzmB directly and efficiently cleave laminB in vitro, in situ in isolated nuclei and in cells loaded with perforin and Gzms, even in the presence of caspase inhibitors. LaminB is cleaved by GzmA at concentrations of 3 nM, but GzmB is 50 times less active. GzmA cuts laminB at R392; GzmB cuts at the caspase VEVD231 site. Characteristic laminB fragments generated by Gzm proteolysis also are observed during CTL lysis, even in the presence of caspase inhibitors or in cells overexpressing bcl-2. Lamins A/C are direct substrates of GzmA, but not GzmB. GzmA and GzmB therefore directly target critical caspase substrates in caspase-resistant cells.     

Binding of two desmin derivatives to the plasma membrane and the nuclear envelope of avian erythrocytes: evidence for a conserved site-specificity in intermediate filament-membrane interactions.

Using solution binding assays, we found that a 45-kDa fragment of desmin, lacking 67 residues from the N terminus, could specifically associate with avian erythrocyte nuclear envelopes but not with plasma membranes from the same cells. It was also observed that a 50-kDa desmin peptide, missing 27 C-terminal residues, retained the ability to bind to both membrane preparations. Displacement experiments with an excess of purified vimentin suggested that the two desmin derivatives were interacting with a previously identified vimentin receptor at the nuclear envelope, the protein lamin B [Georgatos, S. & Blobel, G. (1987) J. Cell Biol. 105, 117-127]. Additional analysis by affinity chromatography confirmed this conclusion. Employing an overlay assay, we demonstrated that the 50-kDa fragment, but not the 45-kDa desmin peptide, was capable of interacting with the plasma membrane polypeptide ankyrin (a known vimentin attachment site), as was intact vimentin. Conversely, the nuclear envelope protein lamin B was recognized by both fragments but not by a chymotryptic peptide composed solely of the helical rod domain of desmin. These data imply that the lamin B-binding site on desmin resides within the 21 residues following its helical rod domain, whereas the ankyrin-associating region is localized within its N-terminal head domain, exactly as in the case of vimentin.     

cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins.

The amino acid sequences deduced from cDNA clones of human lamin A and lamin C show identity between these two lamins except for an extra 9.0-kDa carboxyl-terminal tail that is present only in lamin A. Both lamins A and C contain an alpha-helical domain of approximately 360 residues that shows striking homology to a corresponding alpha-helical rod domain that is the structural hallmark of all intermediate filament proteins. However, the lamin alpha-helical domain is 14% larger than that of the intermediate filament proteins. In addition to the extensive homology to intermediate filament proteins as reported [McKeon, F., Kirschner, M. & Caput, D. (1986) Nature (London) 319, 463-468], a different 82-amino acid residue stretch at the carboxyl terminus of lamin A has been deduced and verified by amino acid sequencing. This region contains sequence homology to amino- and carboxylterminal domains of type I and type II epidermal keratins. Implications of the presence of these and other domains in lamins A and C for the assembly of the nuclear lamina are discussed.     

Bidirectional associations between memory and depression moderated by sex and age: Findings from the CLSA

BackgroundResearch has struggled to understand the temporal relationship between cognition and depression. Some literature suggests that depression may be a risk factor for memory decline, while other work indicates that memory decline may precede depression symptoms. The purpose of this study was to clarify the temporal relationship between memory and depression, examining the moderating role of sex and age.MethodsData were drawn from two time points in the Canadian Longitudinal Study on Aging (CLSA). Memory was measured using a composite of immediate and delayed verbal recall scores, and depressive symptoms were measured using the Center for Epidemiologic Studies Short Depression Scale (CESD-10). Separate cross-lagged panel models (CLPMs) were run based on age (i.e., ages 45–64; ages 65+) and sex (n = 51,338).ResultsResults indicated bidirectional associations between depressive symptoms and memory such that depressive symptoms at baseline predicted memory at follow-up (β= 0.029–0.068, with all p-values <0.01) and memory at baseline predicted depressive symptoms at follow-up (β= 0.025–0.033, with all p-values <0.05). The only exception was in the older female group, where memory did not predict depressive symptoms (β= -0.006, p = 0.543). Depressive symptoms at baseline were a stronger predictor of memory at follow-up than memory at baseline was for depressive symptoms at follow-up in all groups except for older males.FindingsThe findings suggest small but consistent bidirectional associations between depression and memory in almost all sex/age groupings. Depressive symptoms tended to be a stronger predictor of memory than memory was for future depressive symptoms.     

Cross-sectional and prospective associations between abdominal adiposity and proinsulin concentration.

The objective of this study was to investigate the associations of total and abdominal obesity with variation in proinsulin concentration in a Native Canadian population experiencing an epidemic of type 2 diabetes mellitus (DM). Between 1993 and 1995, 728 members of a Native Canadian community participated in a population-based survey to determine the prevalence and risk factors for type 2 DM. Samples for glucose, C-peptide, and proinsulin were drawn after an overnight fast, and a 75-g oral glucose tolerance test was administered. Type 2 DM and impaired glucose tolerance (IGT) were diagnosed using World Health Organization criteria. Height, weight, waist circumference, and percent body fat were measured. In 1998, 95 individuals who, at baseline, had IGT or normal glucose tolerance with an elevated 2-h glucose level (> or = 7.0 mM) participated in a follow-up evaluation using the same protocol. After adjustment for age, sex, C-peptide concentration, per cent body fat, and waist circumference, proinsulin was found to be significantly elevated in diabetic subjects, relative to subjects with both impaired and normal glucose tolerance (both P < 0.0001); and the concentration in those with IGT was higher, compared with normals (P < 0.0001). Among nondiabetic subjects, proinsulin showed significant univariate associations with percent body fat, body mass index, and waist circumference (r = 0.34, 0.45, 0.41, respectively, all P < 0.0001). After adjustment for body fat and other covariates, waist circumference remained significantly associated with proinsulin concentration in nondiabetic subjects (r = 0.20, P < 0.0001). In prospective analysis, adjusted for covariates (including baseline IGT and follow-up glucose tolerance status), baseline waist circumference was positively associated with both follow-up and change in proinsulin concentration (r = 0.27, P = 0.01; r = 0.24, P = 0.03, respectively). These data highlight the detrimental effects of abdominal obesity on beta-cell function, and support the hypothesis that beta-cell dysfunction occurs early in the natural history of glucose intolerance.     

Coronary artery disease severity modifies associations between glycemic control and both mortality and myocardial infarction

Aims

This study examined whether the association between hemoglobin A1c (HbA1c) and short-term clinical outcomes is moderated by CAD severity.

Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering.

We have examined the functional consequences of mutations present in defective alleles of the retinoblastoma susceptibility gene (RB1) isolated from two spontaneously arising tumors. Unlike cDNA clones expressing the wild-type protein p110Rb, those encoding the two mutant proteins failed to induce the appearance of senescent cells in transfected Saos-2 human osteosarcoma cells. The mutant proteins were also defective in binding to the E1A oncoprotein, were unable to become hyperphosphorylated, and failed to become tightly associated with nuclear structures. We conclude that mutations in two distinct regions of the protein concomitantly affect these four aspects of p110Rb function.     

Dynamic interplay between nitration and phosphorylation of tubulin cofactor B in the control of microtubule dynamics

Tubulin cofactor B (TCoB) plays an important role in microtubule dynamics by facilitating the dimerization of α- and β-tubulin. Recent evidence suggests that p21-activated kinase 1 (Pak1), a major signaling nodule in eukaryotic cells, phosphorylates TCoB on Ser-65 and Ser-128 and plays an essential role in microtubule regrowth. However, to date, no upstream signaling molecules have been identified to antagonize the functions of TCoB, which might help in maintaining the equilibrium of microtubules. Here, we discovered that TCoB is efficiently nitrated, mainly on Tyr-64 and Tyr-98, and nitrated-TCoB attenuates the synthesis of new microtubules. In addition, we found that nitration of TCoB antagonizes signaling-dependent phosphorylation of TCoB, whereas optimal nitration of TCoB requires the presence of functional Pak1 phosphorylation sites, thus providing a feedback mechanism to regulate phosphorylation-dependent MT regrowth. Together these findings identified TCoB as the third cytoskeleton protein to be nitrated and suggest a previously undescribed mechanism, whereby growth factor signaling may coordinately integrate nitric oxide signaling in the regulation of microtubule dynamics.