Mutant Gene Products Research Articles

How do nematodes transfer phosphorylcholine to carbohydrates?

An unusual aspect of the biology of nematodes is the attachment of phosphorylcholine (PC) to carbohydrate. The attachment appears to play an important role in nematode development and, in some parasitic species, in immunomodulation. This article considers the nature of the biosynthetic pathway of nematode PC-containing glycoconjugates and, in particular, the identity of the final component in the pathway - the enzyme that transfers PC to carbohydrate (the 'PC transferase'). We offer the opinion that the PC transferase could be a member of the fukutin family (fukutin refers to the mutated gene product that causes Fukuyama congenital muscular dystrophy), a group of enzymes with apparent phosphoryl-ligand transferase activity that are found in organisms ranging from bacteria to humans.

Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases.

Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-L-arginyl-poly-L-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA(6308)) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA(6308) and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA(6308)Delta1, which was truncated by one amino acid at the C terminus; point mutated CphA(6308)C595S; and the combined double-mutant CphA(6308)Delta1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA(6308) (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA(6308)Delta2) or three (CphA(6308)Delta3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA(6308). In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA(6308)Delta1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26 degrees C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308) and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308)Delta1.

Inherited Failure Syndromes.

Abstract SCI-10Global and lineage-restricted bone marrow failure syndromes can be acquired or inherited. Each of the known inherited disorders was initially described by observant clinicians before the field had access to modern tools of molecular biology and genetics. Consequently until recently, diagnosis had depended entirely on a clinical context drawn from the medical and family history, careful physical examinations and a few laboratory tests, none of which were pathognomonic. Today, many of the mutated genes responsible for these phenotypes have been identified and diagnostic tests of good reliability are emerging. This presentation will review the advantages and pitfalls associated with the application of newer diagnostic tests for many of these diseases. The molecular genetic insights have provided additional clinically relevant lessons. For example, the diseases are not limited to patients with the “classic” phenotype and can be diagnosed initially in adulthood. A substantial fraction of patients with Fanconi anemia (FA), for example, does not exhibit the cutaneous or skeletal manifestations of the disease. Some FA patients have entirely normal hematopoiesis, including the responses of hematopoietic cells to mitomycin C or diepoxybutane (abnormalities of which are considered to be the diagnostic standard for this disease). Such patients are “mosaics” in which a single hematopoietic stem cell has corrected the defect on one mutant FA allele and gives it and its progeny such a competitive advantage that they repopulate the entire marrow. Establishing the diagnosis in such cases is essential because the patients remain at high risk for squamous cell carcinoma and because their non-hematopoietic cells remain hypersensitive to cross-linking agents. Between these syndromes there are some shared genetic dysfunctions. For example, cells from patients with dyskeratosis congenita, Shwachman-Diamond syndrome, and Diamond-Blackfan anemia have genetic lesions that directly perturb ribosome biogenesis. Few of these disorders are caused by the inactivation of only one gene. There are at least 13 FA genes, more than 6 dyskeratosis congenita genes, more than one Shwachman-Diamond gene, more than 6 Diamond-Blackfan genes, and at least 7 genes for congenital neutropenia. Not surprisingly, some of these genes encode mutually interacting proteins (the most widely studied of these form a nuclear Fanconi interactome). While the identification of involved genes has advanced our levels of diagnostic certainty, the discoveries have posed many challenges and the list of unanswered questions is growing longer. The most cost-effective approaches to diagnosis are not perfectly defined and although mutation analysis is of profound research importance and required for certain management strategies (e.g. in vitro fertilization and pre-implantation genetic diagnosis) the clinical value of assigning patients to specific complementation groups or mutations has not yet been clearly demonstrated. Leading research questions for hematologists focusing on these disorders include: How do these disparate genetic lesions influence the function of hematopoietic stem cells so profoundly? What environmental influences play a role in marrow failure progression and do the mutant gene products interact biochemically with signals evolving from environmental cues? What accounts for the high relative-risk of myelodysplasia and acute leukemia in all of these disorders? Will effective gene therapy reduce the relative risk of MDS and AML? Hypotheses centering on each of these points are now being tested in a number of laboratories and some of them will be summarized in this presentation. DisclosuresNo relevant conflicts of interest to declare.

The unfolded protein response and its relevance to connective tissue diseases

The unfolded protein response (UPR) has evolved to counter the stresses that occur in the endoplasmic reticulum (ER) as a result of misfolded proteins. This sophisticated quality control system attempts to restore homeostasis through the action of a number of different pathways that are coordinated in the first instance by the ER stress-senor proteins IRE1, ATF6 and PERK. However, prolonged ER-stress-related UPR can have detrimental effects on cell function and, in the longer term, may induce apoptosis. Connective tissue cells such as fibroblasts, osteoblasts and chondrocytes synthesise and secrete large quantities of proteins and mutations in many of these gene products give rise to heritable disorders of connective tissues. Until recently, these mutant gene products were thought to exert their effect through the assembly of a defective extracellular matrix that ultimately disrupted tissue structure and function. However, it is now becoming clear that ER stress and UPR, because of the expression of a mutant gene product, is not only a feature of, but may be a key mediator in the initiation and progression of a whole range of different connective tissue diseases. This review focuses on ER stress and the UPR that characterises an increasing number of connective tissue diseases and highlights novel therapeutic opportunities that may arise.

Expression and molecular dynamics studies on effect of amino acid substitutions at Arg344 in human cathepsin A on the protein local conformation

Human lysosomal protective protein/cathepsin A (CathA) is a multifunctional protein that exhibits not only protective functions as to lysosomal glycosidases, i.e., neuraminidase 1 (NEU1) and β-galactosidase (GLB), but also its own serine carboxypeptidase activity, and exhibits conserved structural similarity to yeast and wheat homologs (CPY and CPW). Our previous study revealed that the R344 (Arg344) residue in CathA could contribute to the binding and recognition of the serine peptidase inhibitor chymostatin. We examined here the effects of substitution of R344 with other amino acids, including A, D, E, G, I, K, M, N, P, Q, S, and V, denoted as R344X, including the wild-type CathA, on expression of CathA activity and intracellular processing. Among the mutant gene products, the 54-kDa precursor/zymogen with the R344D substitution was not processed to the 32/20-kDa mature form with CathA activity in a fibroblastic cell line derived from a galactosialidosis patient. Molecular dynamics (MD) simulations on the total twelve R344X mutants and the wild-type revealed that only R344D takes on a significantly different conformation of S293–D295 in the excision peptide (M285–R298) compared to the other R344X mutants; the side chains of S293 and D295 in R344D are exposed on the molecular surface, although those in the other twelve R344X mutants are buried inside the protein. The results of the current work strongly suggest that the distinct conformational change of the S293–D295 region in the R344D protein causes the processing defect of the 54-kDa precursor of the R344D mutant gene product in cultured cells.

Adaptive Sugar Provisioning Controls Survival of C. elegans Embryos in Adverse Environments

The ability to adapt to changing environmental conditions is essential to the fitness of organisms. In some cases, adaptation of the parent alters the offspring's phenotype [1-10]. Such parental effects are adaptive for the offspring if the future environment is similar to the current one but can be maladaptive otherwise [11]. One mechanism by which adaptation occurs is altered provisioning of embryos by the parent [12-16]. Here we show that exposing adult Caenorhabditis elegans to hyperosmotic conditions protects their offspring from these conditions but causes sensitivity to anoxia exposure. We show that this alteration of survival is correlated with changes in the sugar content of adults and embryos. In addition, mutations in gene products that alter sugar homeostasis also alter the ability of embryos to survive in hyperosmotic and anoxic conditions and engage in the adaptive parental effect. Our results indicate that there is a physiological trade-off between the presence of glycerol, which protects animals from hyperosmotic conditions, and glycogen, which is consumed during anoxia. These two metabolites play an essential role in the survival of worms in these adverse environments, and the adaptive parental effect we describe is mediated by the provisioning of these metabolites to the embryo.

Association of HTRA1 Mutations and Familial Ischemic Cerebral Small-Vessel Disease

The genetic cause of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), which is characterized by ischemic, nonhypertensive, cerebral small-vessel disease with associated alopecia and spondylosis, is unclear. In five families with CARASIL, we carried out linkage analysis, fine mapping of the region implicated in the disease, and sequence analysis of a candidate gene. We also conducted functional analysis of wild-type and mutant gene products and measured the signaling by members of the transforming growth factor beta (TGF-beta) family and gene and protein expression in the small arteries in the cerebrum of two patients with CARASIL. We found linkage of the disease to the 2.4-Mb region on chromosome 10q, which contains the HtrA serine protease 1 (HTRA1) gene. HTRA1 is a serine protease that represses signaling by TGF-beta family members. Sequence analysis revealed two nonsense mutations and two missense mutations in HTRA1. The missense mutations and one of the nonsense mutations resulted in protein products that had comparatively low levels of protease activity and did not repress signaling by the TGF-beta family. The other nonsense mutation resulted in the loss of HTRA1 protein by nonsense-mediated decay of messenger RNA. Immunohistochemical analysis of the cerebral small arteries in affected persons showed increased expression of the extra domain-A region of fibronectin and versican in the thickened tunica intima and of TGF-beta1 in the tunica media. CARASIL is associated with mutations in the HTRA1 gene. Our findings indicate a link between repressed inhibition of signaling by the TGF-beta family and ischemic cerebral small-vessel disease, alopecia, and spondylosis.

Autophagosome vaccine cross-protects and breaks tumor immunology paradigm: a p62-dependent mechanism? (41.41)

Abstract Vaccination with MCA sarcoma cells protects from a subsequent challenge with the same, but not other MCA sarcomas. This observation of unique tumor-specific protection is a well-established paradigm (Prehn and Main 1957). We postulated that all MCA sarcomas overexpress common mutated gene products with a short half-life (SLiPs), but only the unique tumor-rejection antigens are stable enough to be cross-presented and induce anti-tumor immunity. We recently reported that with proteosomal blockade, SLiPs could be isolated in autophagic-containing (LC-3) vesicles (termed DRibble) that were efficient for cross-presentation of antigens (Li, Y. et al). Here we examined if vaccination with DRibbles from one MCA sarcoma would cross-protect from a challenge with an antigenically distinct MCA sarcoma. While whole tumor cell vaccines provide protection to unrelated tumor in 0 of 9 comparisons, DRibble vaccines provided significant (p<0.05) protection for 8 of 9 tumors (n=10-25 mice/group). Using a model system we showed that SLiPs play a critical role in providing antigen to DRibbles and that siRNA knock down of p62 reduced SLiPs and the capacity of DRibble to stimulate T cells. These results have shaped a model where polyubiquitinated SLiPs spared degradation by the proteasome, are picked up by p62 and delivered to autophagic vesicles. Used as a vaccine, these vesicles can cross-present neo-antigens capable of inducing cross-protection of chemically induced sarcomas. Supported by CA80964 (BAF), CA107243 (HH) the Chiles Foundation and the Murdoch Trust.

Reversing Cancer From Inside and Out: Oncogene Addiction, Cellular Senescence, and the Angiogenic Switch

Cancer is largely caused by genetic events that result in the mutation of oncogenes or tumor suppressor genes, leading to cell autonomous proliferation and growth. The repair of these mutant gene products may be expected to subvert this neoplastic behavior. Indeed, oncogene inactivation can result in the elimination of all or almost all tumor cells by various mechanisms through the phenomena described as oncogene addiction. Recently, we have shown that oncogene addiction occurs through at least two broad classes of mechanisms: tumor cell intrinsic mechanisms of cellular senescence and apoptosis; and tumor cell extrinsic host-dependent mechanisms that include the shut-down of angiogenesis. We have argued that the abatement of oncogenic activity within a cancer cell not only leads to the demise of a tumor from within but also through the instruction of the restoration of the microenvironment.

A Mutation in Rab38 Small GTPase Causes Abnormal Lung Surfactant Homeostasis and Aberrant Alveolar Structure in Mice

The chocolate mutation, which is associated with oculocutaneous albinism in mice, has been attributed to a G146T transversion in the conserved GTP/GDP-interacting domain of Rab38, a small GTPase that regulates intracellular vesicular trafficking. Rab38 displays a unique tissue-specific expression pattern with highest levels present in the lung. The purpose of this study was to characterize the effects of Rab38-G146T on lung phenotype and to investigate the molecular basis of the mutant gene product (Rab38(cht) protein). Chocolate lungs exhibited a uniform enlargement of the distal airspaces with mild alveolar destruction as well as a slight increase in lung compliance. Alveolar type II cells were engorged with lamellar bodies of increased size and number. Hydrophobic surfactant constituents (ie, phosphatidylcholine and surfactant protein B) were increased in lung tissues but decreased in alveolar spaces, consistent with a malfunction in lamellar body secretion and the subsequent cellular accumulation of these organelles. In contrast to wild-type Rab38, native Rab38(cht) proteins were found to be hydrophilic and not bound to intracellular membranes. Unexpectedly, recombinant Rab38(cht) proteins retained GTP-binding activity but failed to undergo prenyl modification that is required for membrane-binding activity. These results suggest that the genetic abnormality of Rab38 affects multiple lysosome-related organelles, resulting in lung disease in addition to oculocutaneous albinism.

The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F

The retinoblastoma protein pRb plays a pivotal role in G(1)- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH(2)-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb(DeltaCDK) (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb(DeltaCDK) sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH(2)-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH(2)-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbDeltaCDK(+T373) or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH(2)-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

Membrane Traffic and Muscle: Lessons from Human Disease

Like all mammalian tissues, skeletal muscle is dependent on membrane traffic for proper development and homeostasis. This fact is underscored by the observation that several human diseases of the skeletal muscle are caused by mutations in gene products of the membrane trafficking machinery. An examination of these diseases and the proteins that underlie them is instructive both in terms of determining disease pathogenesis and of understanding the normal aspects of muscle biology regulated by membrane traffic. This review highlights our current understanding of the trafficking genes responsible for human myopathies.

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Endothelial Dysfunction and the Pathobiology of Accelerated Atherosclerosis in Hutchinson‐Gilford Progeria Syndrome

A prominent feature of Hutchinson‐Gilford Progeria Syndrome (HGPS) is premature, severe atherosclerosis, resulting in fatal heart attacks and strokes. Given the critical role of endothelial dysfunction in atherogenesis, we explored the effects of progerin (the mutant gene product in HGPS) on endothelial pathobiology, utilizing a model system in which cultured human endothelial cells (EC) express progerin via adenoviral infection. Progerin accumulation in EC results in a strikingly abnormal nuclear morphology, observed as aggregation of progerin and other nuclear proteins. Accumulation of progerin also induces a proinflammatory/atherogenic program of EC activation characterized by 1) sustained expression of leukocyte adhesion molecules (VCAM‐1, E‐selectin) and proinflammatory cytokines (IL‐8, MCP‐1); 2) expression of prothrombotic genes (PAI‐1); 3) decreased expression of eNOS; and 4) downregulation of KLF2, a transcription factor critical to endothelial homeostasis. Further, conditioned medium from progerin‐expressing EC induces dysfunction when transferred onto control EC. Thus, progerin accumulation in EC appears to induce a state of chronic dysfunction, via autocrine/paracrine pathways, potentially contributing to the premature and accelerated atherosclerosis of HGPS.

Separating Genetic and Hemodynamics Effects In Nrp1 Knockout Embryos

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. Since blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the Neuropilin‐1 (Nrp‐1) receptor, and hemodynamic defects occurring in homozygous knockout embryos. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow‐induced deficiencies from those caused by the nrp‐1 mutation, we arrested blood flow in cultured wild‐type and mutant embryos and followed their vascular development. We find that loss of nrp‐1 function rather than flow induces the altered geometry of the capillary plexus. This method allows genetic defects caused by loss‐of‐function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Small Molecule Inhibitor of Neutrophil Elastase Restores Impaired Production of Human Myeloid Cells Observed in Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a rare hematopoietic disease characterized by maturation arrest at the promyelocytes, recurring severe infections, and evolution to leukemia. Heterozygous mutations in either the neutrophil elastase (NE, ELA2) gene (sporadic or autosomal-dominant SCN) or homozygous mutations in the HAX1 gene (autosomal-recessive SCN) are associated with a similar clinical phenotype and similar block of myeloid differentiation in the marrow. Most studies now indicate that the maturation arrest in SCN is due to accelerated apoptosis of myeloid progenitors triggered by the mutant gene products. The hetero and homozygous deletion of NE in mice as well as the knock-in of mutant NE identified in SCN/AML patient failed to produce severe neutropenia phenotype in mice. We established a cellular model of SCN with inducible expression of del.145–152 NE mutant in human promyelocytic tet-off HL60 cells. Ratio of normal /mutant NE products in these cells is approximately 1:1, similar to that expected in SCN patients with heterozygous NE mutation. Expression of mutant NE in promyelocytic cells resulted in a characteristic block of myeloid differentiation with ∼70% decline in differentiated neutrophils which is similar to that observed in SCN. Cell growth reduction and accelerated apoptosis were also observed in these cells in response to mutant NE expression. Thus, this SCN model appears to closely recapitulate the human phenotype. Analysis of the proteolytic activity of cells expressing mutant elastase revealed approximately 40% increase in total NE-specific activity in response to mutant NE expression compared with controls, suggesting that mutant elastase exhibits at least some proteolytic activity. To date we identified more than 40 heterozygous mutations in the NE gene in pre-leukemic SCN patients, however, the pathomechanism remains unclear. Molecular modeling of the NE tertiary structure revealed that these mutations predominantly affect the N-glycosylation sites or the binding pocket of elastase. Importantly, the active site of the mutant protease appears to be intact, which suggested that NE-specific small molecule inhibitors may be useful in preventing accelerated apoptosis and the block of myeloid differentiation of myeloid cells. Examining this SCN model, we identified a proprietary cell-permeable elastase-specific small molecule inhibitor (compound A, Merck, USA), which inhibited the proteolytic activity of the NE by more than 80%. Our studies indicate that treatment of human promyelocytic cells expressing del.145–152 mutant NE with this small molecule inhibitor restored the impaired production of myeloid cells and improved myeloid differentiation to near normal level. Importantly, the compound A did not impair the growth rate of control cells with normal NE expression. These data suggest that NE-specific small molecule inhibitor and its analogs should be considered in clinical trials in patients with SCN that is attributable to mutant NE.

Improved Retinal Transduction In Vivo and Photoreceptor-specific Transgene Expression Using Adenovirus Vectors With Modified Penton Base

Adenovirus (Ad) vectors can be injected into human ocular tissues without producing adverse events and are therefore a promising means of gene transfer to the retina. However, when administered subretinally, Ad vectors primarily transduce the retinal pigment epithelium (RPE), whereas the majority of mutant gene products that cause photoreceptor (PR) degeneration are expressed exclusively in the PR cells. While it has been shown previously that pseudotyping of Ad can partially overcome the limited PR transduction by Ad5, we found that pseudotyping of Ad is not necessary for transduction of PR cells. We determined that, in the context of Ad, the cytomegalovirus (CMV) promoter is not significantly active in PRs. We compared expression levels from CMV and chicken beta actin (CBA) promoters in neural retina and found that CBA has a 173-fold greater potency than CMV. We also investigated the nature of the Ad-RPE interaction in murine retina and determined that the RGD domain in Ad penton plays a key role in RPE tropism. Deletion of the RGD domain coupled with use of the CBA promoter permitted transgene expression in neural retina approximately 667 times more efficiently than with Ad5 vectors. The use of these vectors in combination with a 4.7 kilobase (kb) rhodopsin promoter enabled transgene expression exclusively in PR cells in vivo.

Thematic review series: Adipocyte Biology. Lipodystrophies: windows on adipose biology and metabolism

The lipodystrophies are characterized by loss of adipose tissue in some anatomical sites, frequently with fat accumulation in nonatrophic depots and ectopic sites such as liver and muscle. Molecularly characterized forms include Dunnigan-type familial partial lipodystrophy (FPLD), partial lipodystrophy with mandibuloacral dysplasia (MAD), Berardinelli-Seip congenital generalized lipodystrophy (CGL), and some cases with Barraquer-Simons acquired partial lipodystrophy (APL). The associated mutant gene products include 1) nuclear lamin A in FPLD type 2 and MAD type A; 2) nuclear lamin B2 in APL; 3) nuclear hormone receptor peroxisome proliferator-activated receptor gamma in FPLD type 3; 4) lipid biosynthetic enzyme 1-acylglycerol-3-phosphate O-acyltransferase 2 in CGL type 1; 5) integral endoplasmic reticulum membrane protein seipin in CGL type 2; and 6) metalloproteinase ZMPSTE24 in MAD type B. An unresolved question is whether metabolic disturbances are secondary to adipose repartitioning or result from a direct effect of the mutant gene product. Careful analysis of clinical, biochemical, and imaging phenotypes, using an approach called "phenomics," reveals differences between genetically stratified subtypes that can be used to guide basic experiments and to improve our understanding of common clinical entities, such as metabolic syndrome or the partial lipodystrophy syndrome associated with human immunodeficiency virus infection.

Gene Transfer of Connexin43 Mutants Attenuates Coupling in Cardiomyocytes

Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector-mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.

Autosomal dominant polycystic kidney disease: pathophysiology and treatment

Autosomal dominant polycystic disease (ADPKD) is one of the most common monogenic disorders, and globally is the third most common cause of end-stage kidney disease. Until recently, the causes of this disease remained obscure. However, the past decade has seen enormous advances in the understanding of the pathophysiology and genetics of this condition, and recent studies have suggested the possibility of specific treatment for slowing cyst growth. This review attempts to address three central questions: (i) how cysts are created; (ii) how mutated gene products give rise to cysts; and (iii) the prospects for treatment. Cysts usually develop from the collecting duct,1,,2 and are highly focal in nature, only developing in a very small proportion of nephrons. They generally increase in size and number throughout life, and encroach on normal renal tissue, causing macrophage infiltration, neovascularization, progressive fibrosis and a slow deterioration in renal function.3,,4 The disease only reaches the stage of chronic renal failure or end-stage renal disease in the fourth or fifth decade in the case of autosomal dominant polycystic disease type 1 (PKD1), or a decade later in the case of autosomal dominant polycystic disease type 2 (PKD2),5 in approximately 50% of patients. Cysts are originally connected to the mother tubule, but eventually the connection closes off, and the cysts can then only enlarge by a process of increased proliferation of mural epithelial cells and tubular secretion. In ADPKD, these cells undergo a remarkable phenotypic change, from the usual non-proliferative and reabsorptive phenotype of collecting duct principal cells, to a secretory and proliferative phenotype; this process has been extensively investigated by Grantham and coworkers.6–10 They initially microdissected cysts from polycystic kidneys, and suspended them in a hydrated collagen gel. The growth of the cysts could then be measured by … Address correspondence to Dr J. Rapoport, Department of Nephrology & Hypertension, Kaplan Medical Center, POB 1, Rehovot 76100, Israel. e-mail: jayson_r{at}clalit.org.il