Kristmundur Sigmundsson

Nephrin Promotes Cell-Cell Adhesion through Homophilic Interactions

Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca(2+)concentration, indicating that the binding is not dependent on, but rather promoted by Ca(2+). Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Expression and Signaling in Human, Mouse, and Rat Leukocytes: Evidence for Replacement of the Short Cytoplasmic Domain Isoform by Glycosylphosphatidylinositol-Linked Proteins in Human Leukocytes

Carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1), the primordial carcinoembryonic Ag gene family member, is a transmembrane cell adhesion molecule expressed in leukocytes, epithelia, and blood vessel endothelia in humans and rodents. As a result of differential splicing, CEACAM1 occurs as several isoforms, the two major ones being CEACAM1-L and CEACAM1-S, that have long (L) or short (S) cytoplasmic domains, respectively. The L:S expression ratios vary in different cells and tissues. In addition to CEACAM1, human but not rodent cells express GPI-linked CEACAM members (CEACAM5–CEACAM8). We compared the expression patterns of CEACAM1-L, CEACAM1-S, CEACAM6, and CEACAM8 in purified populations of neutrophilic granulocytes, B lymphocytes, and T lymphocytes from rats, mice, and humans. Human granulocytes expressed CEACAM1, CEACAM6, and CEACAM8, whereas human B lymphocytes and T lymphocytes expressed only CEACAM1 and CEACAM6. Whereas granulocytes, B cells, and T cells from mice and rats expressed both CEACAM1-L and CEACAM1-S in ratios of 2.2–2.9:1, CEACAM1-S expression was totally lacking in human granulocytes, B cells, and T cells. Human leukocytes only expressed the L isoforms of CEACAM1. This suggests that the GPI-linked CEACAM members have functionally replaced CEACAM1-S in human leukocytes. Support for the replacement hypothesis was obtained from experiments in which the extracellular signal-regulated kinases (Erk)1/2 were activated by anti-CEACAM Abs. Thus, Abs against CEACAM1 activated Erk1/2 in rat granulocytes, but not in human granulocytes. Erk1/2 in human granulocytes could, however, be activated by Abs against CEACAM8. We demonstrated that CEACAM1 and CEACAM8 are physically associated in human granulocytes. The CEACAM1/CEACAM8 complex in human cells might accordingly play a similar role as CEACAM1-L/CEACAM1-S dimers known to occur in rat cells.

The structure of p53 tumour suppressor protein reveals the basis for its functional plasticity

p53 major tumour suppressor protein has presented a challenge for structural biology for two decades. The intact and complete p53 molecule has eluded previous attempts to obtain its structure, largely due to the intrinsic flexibility of the protein. Using ATP‐stabilised p53, we have employed cryoelectron microscopy and single particle analysis to solve the first three‐dimensional structure of the full‐length p53 tetramer (resolution 13.7 Å). The p53 molecule is a D2 tetramer, resembling a hollow skewed cube with node‐like vertices of two sizes. Four larger nodes accommodate central core domains, as was demonstrated by fitting of its X‐ray structure. The p53 monomers are connected via their juxtaposed N‐ and C‐termini within smaller N/C nodes to form dimers. The dimers form tetramers through the contacts between core nodes and N/C nodes. This structure revolutionises existing concepts of p53s molecular organisation and resolves conflicting data relating to its biochemical properties. This architecture of p53 in toto suggests novel mechanisms for structural plasticity, which enables the protein to bind variably spaced DNA target sequences, essential for p53 transactivation and tumour suppressor functions.

RETRACTED: Vulnerability of Glioblastoma Cells to Catastrophic Vacuolization and Death Induced by a Small Molecule

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.

Mechanism of Chain Selection in the Assembly of Collagen IV A PROMINENT ROLE FOR THE α2 CHAIN

Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the α1(IV) and α2(IV) NC1 domains. We show that the differential affinity of α2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the α1(IV) NC1 domain, we conclude that the α2(IV) chain plays a regulatory role in directing chain composition in the assembly of (α1)2α2 triple-helical molecule. Detailed crystal structure analysis of the [(α1)2α2]2 NC1 hexamer and sequence alignments of the NC1 domains of all six α-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a β-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.

The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters

Structural analyses reveal that oligomerization between cell adhesion molecules in the same membrane is influenced by their interactions across opposing membranes (see also in this issue the accompanying paper by Müller et al.).

Computational Analysis of Isoform-Specific Signal Regulation by CEACAM1—A Cell Adhesion Molecule Expressed in PC12 Cells

Abstract: CEACAM1 is a signal‐regulating, homophilic cell adhesion receptor system expressed in epithelia, vessel endothelia, and leukocytes. Here, we demonstrate that CEACAM1 is expressed also in PC12 cells, both as the common transmembrane isoforms, CEACAM1‐L and CEACAM1‐S, and as a novel, secreted, differentially spliced isoform. CEACAM1 can have both positive and negative effects on cell signaling. In an attempt to explain this dual behavior, we have initiated computational analysis of the signal‐regulating effects of CEACAM1. This suggests that CEACAM1 can exert its signal‐regulating activities by discriminating between binding of Src kinases and SHP phosphatases, respectively. Major factors that regulate this discrimination are the expression levels and expression ratios of transmembrane CEACAM1‐L and CEACAM1‐S, the concentration of secreted CEACAM1, and homophilic binding of CEACAM1 presented by neighboring cells.

Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1

The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.

The cell adhesion molecule C-CAM is a substrate for tissue transglutaminase

C‐CAM, a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family, appears as two co‐expressed isoforms, C‐CAM‐L and C‐CAM‐S, with different cytoplasmic domains, that can form homo‐dimers in epithelial cells. In addition, C‐CAM‐L has been found in large molecular weight forms suggesting posttranslational, covalent modification. Here we have investigated the possibility that the cytoplasmic domain of C‐CAM‐L can act as a transglutaminase substrate. Glutathione S‐transferase fusion proteins of the cytoplasmic domains of rat and mouse C‐CAM‐L as well as free cytoplasmic domains, released by thrombin cleavage from the fusion proteins, were converted into covalent dimers by tissue transglutaminase. These results demonstrate that the cytoplasmic domains of rat and mouse C‐CAM‐L are substrates for tissue transglutaminase, and lend support to the notion that higher molecular weight forms of C‐CAM‐L are formed by transglutaminase modification.

Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Arylsulfonamides

The inhibition of insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) by angiotenesin IV is known to improve memory and learning in rats. Screening 10 500 low-molecular-weight compounds in an enzyme inhibition assay with IRAP from Chinese Hamster Ovary (CHO) cells provided an arylsulfonamide (N-(3-(1H-tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfonamide), comprising a tetrazole in the meta position of the aromatic ring, as a hit. Analogues of this hit were synthesized, and their inhibitory capacities were determined. A small structure–activity relationship study revealed that the sulfonamide function and the tetrazole ring are crucial for IRAP inhibition. The inhibitors exhibited a moderate inhibitory potency with an IC50=1.1±0.5 μm for the best inhibitor in the series. Further optimization of this new class of IRAP inhibitors is required to make them attractive as research tools and as potential cognitive enhancers.