Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and a-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigo

Role of the C-terminal extensions of α-Crystallins

Several small heat shock proteins contain a well conserved α-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of αA- and αB-crystallins, is not well understood. We have swapped the C-terminal extensions between αA- and αB-crystallins and generated two novel chimeric proteins, αABc and αBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric αB with the C-terminal extension of αA-crystallin, αBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric αA with the C-terminal extension of αB-crystallin, αABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that αBAc exhibits more solvent-exposed hydrophobic pockets than αA, αB, or αABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of αBAc differs from that of αB-crystallin whereas that of αABc overlaps with that of αA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function

The IXI/V motif in the C-terminal extension of α-crystallins: alternative interactions and oligomeric assemblies

Purpose: α-Crystallin, a hetero-oligomer of αA- and αB-crystallin, is involved in maintaining eye lens transparency, primarily by its structural packing and chaperone activity. αA- and αB-crystallin share significant sequence homology, which is almost exclusively restricted to the central, conserved "αA-crystallin domain". The flanking N-terminal domain and C-terminal extension are highly variable both in sequence and length. Mutations and age-related post-translational modifications of these proteins are associated with congenital and age-onset cataracts. Interestingly, most mutations or truncations in the C-terminal extensions of the α-crystallins and other α-sHsps like Hsp27 lead to pathology. It is therefore important to understand the structure/function relationship of this region. Sequence alignment of the C-terminal extensions of αA- and αB-crystallin with other homologues shows a conserved IXI/V motif. The purpose of this study was to investigate the role of this conserved motif, IPV in αA-crystallin and IPI in αB-crystallin (corresponding to residues 159-161 in both crystallins), in the structure and chaperone activity. Methods: The isoleucine/valine residues in the IPV motif of αA-crystallin and the IPI motif of αB-crystallin were mutated to glycine and studied the secondary and tertiary structure of the mutant proteins using circular dichroism and fluorescence spectroscopy, and the quaternary structure using glycerol density gradient centrifugation and dynamic light scattering. Chaperone activity was studied at 37 °C and 25 °C using DTT induced aggregation of insulin as a model system. We have performed fluorescence resonance energy transfer (FRET) experiments to investigate the interactions of this motif in homo- and hetero-oligomers. Since αB-crystallin is devoid of Cys residues, we have introduced a Cys residue flanking the IPI motif (T162CαB-crystallin) to facilitate fluorescence labeling studies. Results: Unlike in other homologues from plants or prokaryotes, mutation of the isoleucine/valine residues in α-crystallins does not result in oligomer dissociation or loss of chaperone activity. On the contrary, the mutant proteins retain their capacity to oligomerize and show enhanced chaperone activity at 37 °C. The mutants also exhibit significantly higher chaperone-like activity at 25 °C. FRET experiments show that the region spanning the IPI/V motif comes in proximity either to the β-strands of the "α-crystallin" domain or the corresponding IPI/V region of another subunit. Conclusions: Our mutational studies show that the IPI/V motif has a propensity to participate in inter-subunit interactions, either with regions in the α-crystallin domain or with the corresponding IPI/V region on another monomer. These interactions are important in the structure and function of α-crystallins. This motif also appears to be important in the temperature dependent chaperone-like activity of the α-crystallins. The propensity of the IPI/V motif to form multiple inter-subunit interactions may contribute to the diversity in structure and function seen in the α-crystallin/sHsp family

Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome

Smith Lemli Opitz syndrome (SLOS) is an inherited malformation and mental retardation metabolic disorder with no cure. Mutations in the last enzyme of the cholesterol biosynthetic pathway, 7-dehydrocholesterol reductase (DHCR7), lead to cholesterol insufficiency and accumulation of its dehyrdocholesterol precursors, and contribute to its pathogenesis. The central nervous system (CNS) constitutes a major pathophysiological component of this disorder and remains unamenable to dietary cholesterol therapy due to the impenetrability of the blood brain barrier (BBB). The goal of this study was to restore sterol homeostasis in the CNS. To bypass the BBB, gene therapy using an adeno-associated virus (AAV-8) vector carrying a functional copy of the DHCR7 gene was administered by intrathecal (IT) injection directly into the cerebrospinal fluid of newborn mice. Two months post-treatment, vector DNA and DHCR7 expression was observed in the brain and a corresponding improvement of sterol levels seen in the brain and spinal cord. Interestingly, sterol levels in the peripheral nervous system also showed a similar improvement. This study shows that IT gene therapy can have a positive biochemical effect on sterol homeostasis in the central and peripheral nervous systems in a SLOS animal model. A single dose delivered three days after birth had a sustained effect into adulthood, eight weeks post-treatment. These observations pave the way for further studies to understand the effect of biochemical improvement of sterol levels on neuronal function, to provide a greater understanding of neuronal cholesterol homeostasis, and to develop potential therapies

Mechanism and Substrate Recognition of Human Holo ACP Synthase

Mammals utilize a single phosphopantetheinyl transferase for the posttranslational modification of at least three different apoproteins: the carrier protein components of cytosolic and mitochondrial fatty acid synthases and the aminoadipate semialdehyde reductase involved in lysine degradation. We determined the crystal structure of the human phosphopantetheinyl transferase, a eukaryotic phosphopantetheinyl transferase characterized, complexed with CoA and Mg2+, and in ternary complex with CoA and ACP. The involvement of key residues in ligand binding and catalysis was confirmed by mutagenesis and kinetic analysis. Human phosphopantetheinyl transferase exhibits an α/β fold and 2-fold pseudosymmetry similar to the Sfp phosphopantetheinyl transferase from Bacillus subtilis. Although the bound ACP exhibits a typical four-helix structure, its binding is unusual in that it is facilitated predominantly by hydrophobic interactions. A detailed mechanism is proposed describing the substrate binding and catalytic process

αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid β-peptide and β(2)-microglobulin

αB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of αB-crystallin on the fibril growth of the Aβ (amyloid β)-peptides Aβ-(1–40) and Aβ-(1–42). αB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Aβ-(1–40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric α-crystallins in preventing Aβ-(1–40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on α-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Aβ-(1–40). αB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Aβ-(1–42), as well as the fibril growth of Aβ-(1–40) when seeded with the Aβ-(1–42) fibril seed. Sedimentation velocity measurements show that αB-crystallin does not form a stable complex with Aβ-(1–40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. αB-crystallin binds to the amyloid fibrils of Aβ-(1–40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that αB-crystallin prevents the fibril growth of β(2)-microglobulin under acidic conditions. It also retards the depolymerization of β2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimer's disease

Compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein lipoylation and energy disequilibrium.

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor

Characterization of anemic KO mice.

<p>Approximately 20% of the KO mice were found to be anemic. A–E: blood cell analysis. A: Red blood cell distribution width. Anemic KO mice (5 males and 5 females) were distinguished from non-anemic KO (20 males and 11 females) and HF control (10 males, 9 females) on the basis of lower hematocrit (panel B), lower hemoglobin level (panel C), enlarged red cells (panel D) and elevated reticulocyte levels (panel E). **Significantly different (p<0.05) from non-anemic KO and HF control mice; *significantly different from HF controls. None of the parameters were significantly different between male and female KOs. No major differences were seen in other blood cell components. The complete blood count analysis is presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047196#pone.0047196.s005" target="_blank">Table S1</a>. F: Wright's-stained blood smears from anemic KO and HF control male mice. Green arrows indicate enlarged erythrocytes, black arrows codocytes. G: Rectal prolapse in KO female, 9 months post-induction; 9 of the 10 anemic mice exhibited rectal prolapse. H. Blood spots on cage floor blot (∼9″×6″) obtained by 5 min exposure to 3 anemic mice with rectal prolapse; no blood spots were observed on blots from cages housing non-anemic KO or HF mice. I: Red cell turnover study performed on 2 anemic KOs and 4 HF control female mice. The two anemic mice had reticulocyte levels of 13.4 and 20.2%. J: Plasma bilirubin levels in HF (n = 6), non-anemic KO (n = 5) and anemic KO (n = 2) mice; differences between groups were not statistically significant.</p

Genotype and phenotype of the <i>Mcat</i> knockout mouse.

<p>A–D: genotyping by PCR. Mice were screened by PCR analysis of tail DNA, using the primer pair p5:pEx2, which generates 900 and 300 bp products from the floxed and wild-type alleles, respectively (panels A and D), and those carrying the floxed <i>Mcat</i> allele were bred to homozygosity (HF). HF mice were mated with <i>Cre</i> mice and bred to homozygosity for the HF <i>Mcat</i> allele and hemizygosity for the <i>Cre</i> gene, to give the KO genotype. The <i>Cre</i> gene was detected by the unique 100 bp product amplified in the <i>Cre</i>-PCR reaction, in addition to the 300 bp internal positive control (panel B). After treatment with tamoxifen, deletion of exon 2 was detected in various tissues from the KO mice, as judged by two independent PCR reactions (panels C and D). Primer pair p5:pEx2 detects the HF allele (900 bp product) and primer pair p5:p3 generates an 1800 bp product from the HF allele and an 800 bp product from the exon-2-deleted KO allele. Similar results, confirming exon 2 deletion, were observed in skin, bone marrow and lung from KO mice. E–O: phenotyping. E–H, mouse photographs. E and F, Female HF control and KO, respectively, 4 months after tamoxifen treatment. G: Three KO and one HF control female mice 9 months after tamoxifen treatment. H: Male KO mouse 9 months after tamoxifen treatment.</p