Structure of the Nidogen Binding LE Module of the Laminin γ1 Chain in Solution

The structure of the single LE module between residues 791 and 848 of the laminin γ1 chain, which contains the high affinity binding site for nidogen, has been probed using NMR methods. The module folds into an autonomous domain which has a stable and unique three-dimensional (3D) structure in solution. The 3D structure was determined on the basis of 362 interproton distance constraints derived from nuclear Overhauser enhancement measurements and 39 π angles, supplemented by 5 ψ and 22 χ₁angles. The main features of the NMR structures are two-stranded antiparallel β-sheets which are separated by loops and cross-connected by four disulfide bridges. The N-terminal segment which contains the first three disulfide bridges is similar to epidermal growth factor. The C-terminal segment has an S-like backbone profile with a crossover at the last disulfide bridge and comprises two three-residue long β-strands that form an antiparallel β-sheet. The LE module possesses an exposed nidogen binding loop that projects away from the main body of the protein. The side-chains of three amino acids which are crucial for binding (Asp, Asn, Val) are all exposed at the domain surface. An inactivating Asn-Ser mutation in this region showed the same 3D structure indicating that these three residues, and possibly an additional Tyr in an adjacent loop, provide direct contacts in the interaction with nidogen.     

Distinct Roles for Laminin Globular Domains in Laminin α1 Chain Mediated Rescue of Murine Laminin α2 Chain Deficiency

Background

Laminin α2 chain mutations cause congenital muscular dystrophy with dysmyelination neuropathy (MDC1A). Previously, we demonstrated that laminin α1 chain ameliorates the disease in mice. Dystroglycan and integrins are major laminin receptors. Unlike laminin α2 chain, α1 chain binds the receptors by separate domains; laminin globular (LG) domains 4 and LG1-3, respectively. Thus, the laminin α1 chain is an excellent tool to distinguish between the roles of dystroglycan and integrins in the neuromuscular system.

Methodology/Principal Findings

Here, we provide insights into the functions of laminin α1LG domains and the division of their roles in MDC1A pathogenesis and rescue. Overexpression of laminin α1 chain that lacks the dystroglycan binding LG4-5 domains in α2 chain deficient mice resulted in prolonged lifespan and improved health. Importantly, diaphragm and heart muscles were corrected, whereas limb muscles were dystrophic, indicating that different muscles have different requirements for LG4-5 domains. Furthermore, the regenerative capacity of the skeletal muscle did not depend on laminin α1LG4-5. However, this domain was crucial for preventing apoptosis in limb muscles, essential for myelination in peripheral nerve and important for basement membrane assembly.

Conclusions/Significance

These results show that laminin α1LG domains and consequently their receptors have disparate functions in the neuromuscular system. Understanding these interactions could contribute to design and optimization of future medical treatment for MDC1A patients.     

Crystal Structures of the Network-Forming Short-Arm Tips of the Laminin β1 and γ1 Chains

The heterotrimeric laminins are a defining component of basement membranes and essential for tissue formation and function in all animals. The three short arms of the cross-shaped laminin molecule are composed of one chain each and their tips mediate the formation of a polymeric network. The structural basis for laminin polymerisation is unknown. We have determined crystal structures of the short-arm tips of the mouse laminin β1 and γ1 chains, which are grossly similar to the previously determined structure of the corresponding α5 chain region. The short-arm tips consist of a laminin N-terminal (LN) domain that is attached like the head of a flower to a rod-like stem formed by tandem laminin-type epidermal growth factor-like (LE) domains. The LN domain is a β-sandwich with elaborate loop regions that differ between chains. The γ1 LN domain uniquely contains a calcium binding site. The LE domains have little regular structure and are stabilised by cysteines that are disulphide-linked 1-3, 2-4, 5-6 and 7-8 in all chains. The LN surface is not conserved across the α, β and γ chains, but within each chain subfamily there is a striking concentration of conserved residues on one face of the β-sandwich, while the opposite face invariably is shielded by glycans. We propose that the extensive conserved patches on the β and γ LN domains mediate the binding of these two chains to each other, and that the α chain LN domain subsequently binds to the composite β-γ surface. Mutations in the laminin β2 LN domain causing Pierson syndrome are likely to impair the folding of the β2 chain or its ability to form network interactions.     

Chemical properties of guinea-pig immunoglobulins γ2G and γM

The isolation of guinea-pig immunoglobulins γ₁G, γ₂G and γM are described and methods for separating the polypeptide chains of each examined. The molecular weights, extinction coefficients and carbohydrate and amino acid compositions of the immunoglobulins and their constituent chains have been analysed. The findings provide a basis for further studies attempting to relate structural differences to distinct biological properties of guinea-pig immunoglobulins.     

Crystal Structure of the LG1-3 Region of the Laminin ��2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function. Their essential roles in embryo development and tissue function have been demonstrated by numerous genetic studies and the analysis of severe human diseases resulting from mutations in laminin genes (1–4). All laminins are heterotrimers composed of three different gene products, termed α, β, and γ chains. At present, 16 mouse and human laminins are known, assembled from five α, three β, and three γ chains. The different laminins have characteristic expression patterns and functions in the embryo and adult animal (1). Laminins are cross-shaped molecules: the three short arms are composed of one chain each, while the long arm is a coiled coil of all three chains, terminating in a tandem of five laminin G-like (LG)² domains, LG1-5, contributed by the α chain (2). Basement membrane assembly requires polymerization via the short arms and cell attachment via the LG1-5 region (5, 6).Cell adhesion to laminins is mediated by multiple receptors: integrins bind to the LG1-3 region, whereas α-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids bind predominantly to sites in the LG4-5 pair (7). Integrins are heterodimers with a large extracellular domain consisting of one α and one β chain, which both span the cell membrane and engage in transmembrane signaling (8). Of the 24 mouse and human integrins, the major laminin binding integrins are α3β1, α6β1, α7β1, and α6β4, which have distinct affinities for the different laminin isoforms (9). Although some studies have reported integrin binding or integrin-mediated cell adhesion to isolated LG domains or tandems (10–12), there is strong evidence to suggest that the coiled coil region and an intact γ chain tail are required for full integrin binding to the laminin LG1-3 region (13–18). Compared with integrin binding to collagen and fibronectin, which is understood in atomic detail (19, 20), the laminin-integrin interaction remains poorly characterized in structural terms. We previously determined crystal structures of the LG4-5 region of the laminin α1 and α2 chains and defined their receptor binding sites (21–23). Here, we report the crystal structure of the remainder of the laminin α2 receptor binding region, LG1-3.     

Crystal Structure of Three Consecutive Laminin-type Epidermal Growth Factor-like (LE) Modules of Laminin γ1 Chain Harboring the Nidogen Binding Site

The structure of three consecutive laminin-type EGF-like (LE) modules of mouse laminin γ1 chain, γ1III3-5 (positions 738 to 899), has been determined by multiple isomorphous replacement in a crystal of space groupP6₄22 (a=b=74.57 Å,c=185.11 Å and γ=120°). The crystal structure was refined using restrained crystallographic refinement to an R-factor of 19.72 % for 14,983 independent reflections with intensitiesF_obs> 0 at 2.1 Å resolution, with root mean square deviations of 0.012 Å and 1.690° from ideal bond lengths and bond angles, respectively. The final model consisted of 1179 (non-hydrogen) protein atoms within 162 residues and 119 water molecules. The molecule showed a rod-like structure of about 76 Å length with individual modules twisted relative to each other by about 70°. Each module had the same disulfide bond connections Cys1-Cys3 (loop a), Cys2-Cys4 (loop b), Cys5-Cys6 (loop c) and Cys7-Cys8 (loop d), the first three being identical to epidermal growth factor (EGF). All three LE modules showed little secondary structure which was mainly restricted to loop d, but they differed in several other details of their structure. The interface contacts between the LE modules are based on hydrogen bonds and hydrophobic interactions between the hydrophobic core of loop d of the preceding module and the first cysteine and an exposed residue in loop b of the following module. Module 4 was previously shown to contribute the major nidogen binding site of laminins and site-directed mutagenesis demonstrated a specific binding role for Asp800, Asn802, Val804 and Tyr819 in loops a and c. The side-chains of these four residues are all located on the surface in a linear array and separated by a distance of 17 Å between Tyr819 and Val804. The entire nidogen binding site is stabilizedviamain-chain hydrogen bonds which are in part derived from the link between loops b and c (residues Leu815 and Lys816). The data demonstrate the unique nature of the LE modules and only a remote similarity to EGF. They also indicate that the crucial residues in the binding loops provide direct contacts with nidogen and explain the synergism between loops a and c which is essential for binding.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.     

Quantitation of γH2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks¹. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)². Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy². Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer³. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo^4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.Download video file.^{(284M, mp4)}     

Targeting γ-Herpesvirus 68 Bcl-2-mediated Down-regulation of Autophagy

γ-herpesviruses (γHVs) are common human pathogens that encode homologs of the anti-apoptotic cellular Bcl-2 proteins, which are critical to viral reactivation and oncogenic transformation. The murine γHV68 provides a tractable in vivo model for understanding general features of these important human pathogens. Bcl-X_L, a cellular Bcl-2 homolog, and the murine γHV68 Bcl-2 homolog, M11, both bind to a BH3 domain within the key autophagy effector Beclin 1 with comparable affinities, resulting in the down-regulation of Beclin 1-mediated autophagy. Despite this similarity, differences in residues lining the binding site of M11 and Bcl-X_L dictate varying affinities for the different BH3 domain-containing proteins. Here we delineate Beclin 1 differential specificity determinants for binding to M11 or Bcl-X_L by quantifying autophagy levels in cells expressing different Beclin 1 mutants and either M11 or Bcl-X_L, and we show that a G120E/D121A Beclin 1 mutant selectively prevents down-regulation of Beclin 1-mediated autophagy by Bcl-X_L, but not by M11. We use isothermal titration calorimetry to identify a Beclin 1 BH3 domain-derived peptide that selectively binds to M11, but not to Bcl-X_L. The x-ray crystal structure of this peptide bound to M11 reveals the mechanism by which the M11 BH3 domain-binding groove accommodates this M11-specific peptide. This information was used to develop a cell-permeable peptide inhibitor that selectively inhibits M11-mediated, but not Bcl-X_L-mediated, down-regulation of autophagy.     

Analysis of Nidogen-1/Laminin γ1 Interaction by Cross-Linking,Mass Spectrometry,and Computational Modeling Reveals Multiple Binding Modes

We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2–4, γ1 LEb2–4 N836D, γ1 short arm, and γ1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin γ1 LEb2–4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces.     

The products from papain and pepsin hydrolyses of guinea-pig immunoglobulins γ1G and γ2G

1. The products from papain and pepsin hydrolyses of the guinea-pig immunoglobulins gamma(1)G and gamma(2)G were isolated and characterized with regard to molecular weight, amino acid composition, hexose content and antigenic specificity. 2. Fragments Fab and (Fab')(2) from immunoglobulins gamma(1)G and gamma(2)G have similar electrophoretic and antigenic properties, but show some class-specific differences in amino acid composition. 3. Three Fc fragments were obtained after papain digestion of immunoglobulin gamma(2)G, namely, fragment Fc dimer (mol.wt. 58000), fragment Fc monomer (mol.wt. 29000) and fragment Fc' (mol.wt. 8000). A single crystalline fragment, namely fragment Fc' (mol.wt. 11000), was isolated after papain digestion of immunoglobulin gamma(1)G. 4. Peptic digestion of immunoglobulins gamma(1)G and gamma(2)G releases C-terminal fragments, namely, fragments pFc', of similar molecular weight (13000) but different amino acid compositions and distinct antigenic specificities. 5. Digestion-time studies show that immunoglobulin gamma(1)G is far more susceptible to proteolysis than is immunoglobulin gamma(2)G and suggest that at least a proportion of molecules are split primarily at a site that liberates fragment gamma(1)Fc'.     

Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase

The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.     

Supercritical CO2 extraction of fungal oil containing γ-linolenic acid

Supercritical CO2 has been used to extract an oil containing -linolenic acid (GLA) from Cunninghamella echinulata. The highest oil recovery from dry biomass (26.4%, w/w) and GLA yield (26.1 g/kg biomass) has been achieved at 30 MPa and 50 °C after 180 min using fungal particles smaller than 0.5 mm and mass flow of 50 kg CO2/kg dry biomass. Extractions with hexane/ethanol and chloroform/methanol methods gave less than 90% of the GLA/kg reached with the supercritical CO2 method.     

Viral Bcl-2-Mediated Evasion of Autophagy Aids Chronic Infection of γHerpesvirus 68

γ-herpesviruses (γHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine γ-herpesvirus 68 (γHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant γHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which γHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of γ-herpesviruses.     

The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells.     

Structure of the Hydrolyzed Product (F-2) Released from γ-Polyglutamic Acid by γ-Glutamyl Hydrolase YwtD of Bacillus subtilis

The structure of the hydrolyzed product (F-2) with a molecular mass of about 2 kDa released from γ-polyglutamic acid by the γ-glutamyl hydrolase YwtD of Bacillus subtilis was analyzed. The results showed that F-2 is an optically heterogeneous polymer consisting of D- and L-glutamic acid in an 80:20 ratio with D-glutamic acid on both the N- and C-terminal sides, suggesting that YwtD is an enzyme that cleaves the γ-glutamyl bond between D- and D-glutamic acid recognizing adjacent L-glutamic acid toward the N-terminal region.     

Structure and function of γ-secretase

The γ-secretase complex is a prime target for pharmacological intervention in Alzheimer’s disease and so far drug discovery efforts have yielded a large variety of potent and rather specific inhibitors of this enzymatic activity. However, as γ-secretase is able to cleave a wide variety of physiological important substrates, the real challenge is to develop substrate-specific compounds. Therefore, obtaining structural information about γ-secretase is indispensable. As crystal structures of the complex will be difficult to achieve, applied biochemical approaches need to be integrated with structural information obtained from other intramembrane-cleaving proteases. Here we review current knowledge about the structure and function of γ-secretase and discuss the value of these findings for the mechanistic understanding of this unusual protease.