Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.     

Molecular basis of the regulation of Beclin 1-dependent autophagy by the γ-herpesvirus 68 Bcl-2 homolog M11

γ-Herpesviruses (γHVs), including important human pathogens such as Epstein Barr virus, Kaposi’s sarcoma-associated HV, and the murine γHV68, encode homologs of the anti-apoptotic, cellular Bcl-2 (cBcl-2) to promote viral replication and pathogenesis. The precise molecular details by which these proteins function in viral infection are poorly understood. Autophagy, a lysosomal degradation pathway, is inhibited by the interaction of cBcl-2s with a key autophagy effector, Beclin 1, and can also be inhibited by γHV Bcl 2s. Here we investigate the γHV68 M11-Beclin 1 interaction in atomic detail, using biochemical and structural approaches. We show that the Beclin 1 BH3 domain is the primary determinant of binding to M11 and other Bcl 2s, and this domain binds in a hydrophobic groove on M11, reminiscent of the binding of different BH3 domains to other Bcl-2s. Unexpectedly, regions outside of, but contiguous with, the Beclin 1 BH3 domain also contribute to this interaction. We find that M11 binds to Beclin 1 more strongly than do KSHV Bcl-2 or cBcl-2. Further, the differential affinity of M11 for different BH3 domains is caused by subtle, yet significant, variations in the atomic details of each interaction. Consistent with our structural analysis, we find that Beclin 1 residues L116 and F123, and M11 residue pairs G86+R87 and Y60+L74, are required for M11 to bind to Beclin 1 and down-regulate autophagy. Thus, our results suggest that M11 inhibits autophagy through a mechanism that involves the binding of the Beclin 1 BH3 domain in the M11 hydrophobic surface groove.     

Molecular basis of multiple sulfatase deficiency,mucolipidosis II/III and Niemann–Pick C1 disease — Lysosomal storage disorders caused by defects of non-lysosomal proteins

Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann–Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single sulfatase deficiencies.     

Nucleosomal association and altered interactome underlie the mechanism of cataract caused by the R54C mutation of αA-crystallin

BackgroundαA-crystallin plays an important role in eye lens development. Its N-terminal domain is implicated in several important biological functions. Mutations in certain conserved arginine residues in the N-terminal region of αA-crystallin lead to cataract with characteristic cytoplasmic/nuclear aggregation of the mutant protein. In this study, we attempt to gain mechanistic insights into the congenital cataract caused by the R54C mutation in human αA-crystallin.MethodsWe used several spectroscopic techniques to investigate the structure and function of the wild-type and R54CαA-crystallin. Immunoprecipitation, chromatin-enrichment followed by western blotting, immunofluorescence and cell-viability assay were performed to study the interaction partners, chromatin-association, stress-like response and cell-death caused by the mutant.ResultsAlthough R54CαA-crystallin exhibited slight changes in quaternary structure, its chaperone-like activity was comparable to that of wild-type. When expressed in lens epithelial cells, R54CαA-crystallin exhibited a speckled appearance in the nucleus rather than cytoplasmic localization. R54CαA-crystallin triggered a stress-like response, resulting in nuclear translocation of αB-crystallin, disassembly of cytoskeletal elements and activation of caspase 3, leading to apoptosis. Analysis of the “interactome” revealed an increase in interaction of the mutant protein with nucleosomal histones, and its association with chromatin.ConclusionsThe study shows that alteration of “interactome” and nucleosomal association, rather than loss of chaperone-like activity, is the molecular basis of cataract caused by the R54C mutation in αA-crystallin.General significanceThe study provides a novel mechanism of cataract caused by a mutant of αA-crystallin, and sheds light on the possible mechanism of stress and cell death caused by such nuclear inclusions.     

Primary amenorrhea in two sisters: description of a Mexican family with 17α hydroxylase-17 lyase deficiency caused by arginine - stop mutation

A rare cause of congental adrenal hyperplasia is 17α-hydroxylase deficiency. It results in sexual infantilism, primary amenorrhea in females, pseudohermaphroditism in males, hypertension, and hypokalemia. We studied two female siblings from a rural community in Mexico. The cause of consultation was primary amenorrhea. The proband had low levels of estrogen, progesterone and cortisol. Deoxycorticosterone and corticosterone levels were elevated. The proband was homozygous for a transversion of cytosine to thymine at exon 4 (CGA→TGA), causing a premature stop codon at position 239 (R239X). Analysis of family members showed the presence of this heterozygous mutation in the mother, father and one healthy sibling. In summary, we describe a Mexican family with 17α-hydroxylase deficiency due to R239X mutation.     

Gaseous hydrocarbons associated with black layer induced by the interaction of cyanobacteria and Desulfovibrio desulfuricans†

Black layer is a condition of high-sand-content golf greens that results in a subsurface blackened layer in the sand produced by sulfate-reducing bacteria. Black layer can be the product of an interaction of cyanobacteria and sulfate-reducing bacteria and may or may not be toxic to the grass growing on the sand. The organic byproducts of the cyanobacteria coat and plug the sand thereby creating an anoxic environment for development of the sulfate- reducing bacteria. The present study was initiated to determine the range of gaseous hydrocarbons evolved from black layered sand produced by the interaction of two genera of cyanobacteria, Nostoc and Oscillatoria, and Desulfovibrio desulfuricans. The gaseous hydrocarbons measured included methane, ethane, ethylene, and propylene. In nonblackened sand, Nostoc evolved the highest levels of these gases, Oscillatoria evolved relatively low levels except for propylene, and D. desulfuricans evolved the smallest quantities of the gases. When the cyanobacteria and D. desulfuricans were combined to develop black layered sand some changes occurred in the evolution of the gases. Evolution of the gases from Nostoc + D. desulfuricans decreased or remained the same relative to Nostoc alone, and increased relative to D. desulfuricans alone. Except for propylene evolution, gases from Oscillatoria + D. desulfuricans increased relative to Oscillatoria or D. desulfuricans alone. Propylene evolution from Oscillatoria + D. desulfuricans remained unchanged relative to Oscillatoria alone, but increased relative to D. desulfuricans alone. The gases measured are discussed relative to the organisms observed and the conditions of the study.     

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.     

Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling

The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.     

Conformational Studies of the 313-320 and 313-332 Peptide Fragments Derived from the αIIb Subunit of Integrin Receptor with Molecular Dynamics Simulations

The peptide sequence YMESRADRKLAEVGRVYLFL, derived from 313-332 region of the α_IIb, has been identified as a potent inhibitor of platelet aggregation and fibrinogen binding to α_IIbβ₃. More detailed studies have revealed that the Y₃₁₃MESRADR₃₂₀ sequence is the shortest octapeptide with strong inhibitory activity. This work provides insight of the solution conformation of these peptides, by performing extensive molecular dynamics simulations of 100 ns. The 8mer peptide has no stable conformation in water while the 20mer peptide retains a relative conformational stability. Analysis of side chain orientation of the RAD fragment revealed the synplanar arrangement of guanidinium and β-carboxylic groups providing a framework for explaining the similar biological activity of the two peptides, despite their differences in sequence and conformation.     

Activation of p53-p21 is closely associated with the acquisition of resistance to apoptosis caused by β1-integrin silencing in head and neck cancer cells

The issue of whether aberrant expression of β1-integrin is associated with cancer progression and development of resistance to cytotoxic therapy is of considerable interest. Studies to date have shown that the anchorage-independent survival of cancer is attributed, in part, to epithelial-to-mesenchymal transition (EMT). Here, we have reported a novel alternative mechanism of anchorage-independent survival of cancer cells. Cell lines derived from head and neck cancer patients (AMC-HN-3 and AMC-HN-9) and the well-known EMT cancer cell line, MDA-MB231, were examined. The EMT features of AMC-HN-9 cells were comparable to those of MDA-MB231, whereas AMC-HN-3 cells showed no EMT characteristics. Although the pattern and degree of β1-integrin expression were similar in all three cell lines, sensitivities of the cells to β1-integrin knockdown with small interfering RNA (siRNA) were different. Cancer cells with no EMT features underwent cell death to a more significant extent following β1-integrin silencing than those with EMT. Intriguingly, we observed reactive activation of the p53-p21 pathway after β1-integrin silencing in AMC-HN-9 cells lacking an apparent cell death response. Simultaneous knockdown of wild-type p53 and β1-integrin in this cell line promoted cell death. Our data collectively indicate that β1-integrin-related cell death is closely associated with EMT phenotypes and activation of the p53-p21 pathway is partly involved in the acquisition of resistance to apoptosis induced by β1-integrin silencing. Further clarification of the mechanisms underlying p53 integration with β1-integrin signaling may facilitate the development of novel anti-cancer strategies.     

Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998     

A novel I247T missense mutation in the haptoglobin 2 β-chain decreases the expression of the protein and is associated with ahaptoglobinemia

We have identified a novel base substitution at codon 247 in the -chain of the haptoglobin 2 (Hp²) allele in a Ghanaian with the Hp0 (ahaptoglobinemic) phenotype. The heterozygous TC substitution caused reduced expression of the protein when the mutant was transfected into COS7 cells. The base substitution resulted in a missense change of the non-polar amino acid isoleucine to the polar amino acid threonine at a position in the -chain that is highly conserved among several species. We had previously identified a mutation in the Hp gene promoter region for the same individual, which gives her genotype as –61CHp²/–61CHp²(I247T). Since the –61C mutation also leads to low Hp expression, the genotype represents the first and most definitive ahaptoglobinemic case reported in Africa.     

Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.     

Matrix metalloproteinase-9 deficiency leads to prolonged foreign body response in the brain associated with increased IL-1β levels and leakage of the blood-brain barrier

Matrix metalloproteinases (MMPs) are enzymes with specificity towards extracellular matrix (ECM) components. MMPs, especially MMP-9, have been shown to degrade components of the basal lamina and disrupt the blood-brain barrier (BBB) and thus, contribute to neuroinflammation. In the present study we examined the role of MMP-9 in the foreign body response in the brain. Millipore filters of mixed cellulose ester were implanted into the brain cortex of wild type and MMP-9-null mice for a period of 2 d to 8 wks and the response was analyzed by histology and immunohistochemistry. We observed enhanced and prolonged neuroinflammation in MMP-9-null mice, evidenced by persistence of neutrophils, macrophages/microglia, and reactive astrocytes up to 8 wks post-implantation. In addition, blood vessel density around implants was increased in MMP-9-null mice and detection of mouse serum albumin (MSA) indicated that vessels were leaky. Immunohistochemical and western blot analyses indicated that this defect was associated with the absence of tight junction proteins zonula occludens-1 (ZO-1) and ZO-2 from vessels in proximity to implants. Analysis of brain sections and brain protein extracts revealed that the levels of the pro-inflammatory cytokine interleukin-1β (IL-1β), which is a substrate for MMP-9, were significantly higher in MMP-9-null mice at 8wks post-implantation. Collectively, our studies suggest that increased levels of IL-1β and the delayed repair of BBB are associated with prolongation of the FBR in MMP-9-null mice.     

Fatal mitochondrial myopathy,lactic acidosis,and complex I deficiency associated with a heteroplasmic A→G mutation at position 3251 in the mitochondrial tRNALeu(UUR) gene

A girl, who died at 14 years of age from a rapidly progressive mitochondrial myopathy, was found to be heteroplasmic for a mutation in the mitochondrial tRNALeu(UUR) gene at position 3251. A large proportion of muscle fibres contained accumulations of abnormal mitochondria but no cytochrome c oxidase deficient fibres were present. Polarographic and enzymatic measurements on isolated muscle mitochondria revealed a profound isolated complex I deficiency. A high percentage of mutant mtDNA was found in muscle (94%), fibroblasts (93%), brain (90%), liver (80%), and heart (79%). The family was not available for investigation. For genotype to phenotype correlation studies, we investigated the proportion of mutated mtDNA in single muscle fibres of normal appearance and muscle fibres with accumulations of mitochondria. The proportion of mutant mtDNA was 28% (range < 0.3%–86%) in normal-appearing fibres and 61% (range 15%–88%) in abnormal fibres. The difference in the proportion of mutant mtDNA was highly significant (P < 0.001) between the two groups of fibres.     

A causal C–A mutation in the second exon of GS3 highly associated with rice grain length and validated as a functional marker

Comparative sequencing of GS3, the most important grain length (GL) QTL, has shown that differentiation of rice GL might be principally due to a single nucleotide polymorphism (SNP) between C and A in the second exon. A total of 180 varieties representing a wide range of rice germplasm were used for association analysis between C–A mutation and GL in order to confirm the potential causal mutation. A cleaved amplified polymorphic sequence (CAPS) marker, SF28, was developed based on the C–A polymorphism in the GS3 gene. A total of 142 varieties carried allele C with GL from 6.4 to 8.8 mm, while the remaining 38 varieties carried allele A with GL from 8.8 to 10.7 mm. Twenty-four unlinked SSR markers were selected to genotype 180 varieties for population structure analysis. Population structure was observed when the population was classified to three subpopulations. Average GL of either genotype A or genotype C within japonica among the three subpopulations had no significant difference from that in indica, respectively, although indica rice had longer grains on average than japonica in the 180 varieties. However, genotype C always had longer grain length on average than genotype A among three subpopulations. The mutation could explain 79.1, 66.4 and 34.7% of GL variation in the three subpopulations, respectively. These results clearly confirmed the mutation between C and A was highly associated with GL. The SF28 could be a functional marker for improvement of rice grain length.     

Molecular basis of lipopolysaccharide heterogeneity in Escherichia coli: envelope stress-responsive regulators control the incorporation of glycoforms with a third 3-deoxy-α-D-manno-oct-2-ulosonic acid and rhamnose

Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes. Examination of LPS heterogeneity revealed overlapping control by RpoE σ factor, two-component systems (BasS/R and PhoB/R), and ppGpp. Deletion of RpoE-specific anti-σ factor rseA led to near-exclusive incorporation of glycoforms with the third Kdo linked to Kdo disaccharide. This was accompanied by concomitant incorporation of rhamnose, linked to either the terminal third Kdo or to the second Kdo, depending upon the presence or absence of phosphoethanolamine on the second Kdo with truncation of the outer core. This truncation in ΔrseA was ascribed to decreased levels of WaaR glycosyltransferase, which was restored to wild-type levels, including overall LPS composition, upon the introduction of rybB sRNA deletion. Thus, ΔwaaR contained LPS primarily with Kdo(3) without any requirement for lipid A modifications. Accumulation of a glycoform with Kdo(3) and 4-amino-4-deoxy-l-arabinose in lipid A in ΔrseA required ppGpp, being abolished in a Δ(ppGpp(0) rseA). Furthermore, Δ(waaZ lpxLMP) synthesizing tetraacylated lipid A exhibited synthetic lethality at 21-23°C pointing to the significance of the incorporation of the third Kdo.     

Genetic Analysis of δheld and δuvrd Mutations in Combination with Other Genes in the Recf Recombination Pathway in Escherichia Coli: Suppression of a Ruvb Mutation by a Uvrd Deletion

Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background], we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD and the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and recN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Suprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the δuvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two ``RecF' homologous recombination pathways operating in a recBCsbcB(C) strain background.