Proteostasis Strategies for Restoring α1-Antitrypsin Deficiency

The function of the human proteome is defined by the proteostasis network (PN) (Science 2008;319:916; Science 2010;329:766), a biological system that generates, protects, and, where necessary, degrades a protein to optimize the cell, tissue, and organismal response to diet, stress, and aging. Numerous human diseases result from the failure of proteins to fold properly in response to mutation, disrupting the proteome. In the case of the exocytic pathway, this includes proteostasis components that direct folding, and export of proteins from the endoplasmic reticulum (ER). Included here are serpin deficiencies, a class of related diseases that result in a significant reduction of secretion of serine proteinase inhibitors from the liver into serum. In response to misfolding, variants of the serine protease α1-antitrypsin (α1AT) fail to exit the ER and are targeted for either ER-associated degradation or autophagic pathways. The challenge for developing α1AT deficiency therapeutics is to understand the PN pathways involved in folding and export. Herein, we review the role of the PN in managing the protein fold and function during synthesis in the ER and trafficking to the cell surface or extracellular space. We highlight the role of the proteostasis boundary to define the operation of the proteome (Annu Rev Biochem 2009;78:959). We discuss how manipulation of folding energetics or the PN by pharmacological intervention could provide multiple routes for restoration of variant α1AT function to the benefit of human health

MPTAC Determines APP Fragmentation via Sensing Sulfur Amino Acid Catabolism

Summary: Metabolic disorder has been suggested to underlie Alzheimer’s disease (AD). However, the decisive molecular linkages remain unclear. We discovered that human Molybdopterin Synthase Associating Complex, MPTAC, promotes sulfur amino acid catabolism to prevent oxidative damage from excess sulfur amino acids, which, in turn, advances fatty acid oxidation and acetyl coenzyme A (acetyl-CoA) synthesis. The association of MPTAC with Protein arginine (R) Methyltransferase 5 (PRMT5) complex and small nuclear ribonucleoprotein (SNRP) splicing factors enables SNRPs to sense metabolic states through their methylation. This promotes the splicing fidelity of amyloid precursor protein (APP) pre-mRNA and proper APP fragmentation, abnormalities of which have been observed in the platelets of AD patients. The functions of MPTAC are crucial to maintain expression of drebrin 1, which is required for synaptic plasticity, through prevention from oxidative damage. Thus, adjustment of sulfur amino acid catabolism by MPTAC prevents events that occur early in the onset of AD. : Suganuma et al. show that the regulation of sulfur amino acid catabolism by MPTAC suppresses ROS generation and is crucial for the splicing fidelity of APP mRNA, maintaining synaptic plasticity. Thus, MPTAC prevents events that occur early in the onset of Alzheimer’s disease. Keywords: MPTAC, sulfur amino acid catabolism, APP fragmentation, SAMe, PRMT5, MPT synthase, fatty acid beta-oxidation, amyloid bet

Nedd4-2–Mediated Ubiquitination Facilitates Processing of Surfactant Protein–C

We previously proposed a model of surfactant protein (SP)–C biosynthesis in which internalization of the proprotein from the limiting membrane of the multivesicular body to internal vesicles represents a key step in the processing and secretion of SP-C. To test this hypothesis, alanine mutagenesis of the N-terminal propeptide of SP-C was performed. Adenoviruses encoding mutant proproteins were infected into type II cells isolated from Sftpc−/− mice, and media analyzed for secreted SP-C 24 hours after infection. Mutation of S12PPDYS17 completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of neural precursor cell-expressed developmentally down-regulated (Nedd) 4-like E3 ubiquitin ligases. Purified recombinant glutathione S-transferase–SP-C propeptide (residues 1–35) bound recombinant Nedd4-2 strongly, and Nedd4 weakly; the S12PPDYS17mutation abrogated binding of SP-C to Nedd4-2. Immobilized recombinant Nedd4-2 WW domain captured SP-C proprotein from mouse type II cell lysates; in the reverse pulldown, endogenous SP-C in type II cells was captured by recombinant Nedd4-2. To determine if the interaction of Nedd4-2 and SP-C resulted in ubiquitination, the SP-C proprotein was immunoprecipitated from transiently transfected human embryonic kidney 293 cells, and analyzed by SDS-PAGE/Western blotting with ubiquitin antibody. Two ubiquitinated forms of SP-C were detected; ubiquitination was blocked by mutation of K6, but not K34, in the SP-C propeptide. Mutation of K6 also inhibited processing of SP-C proprotein to the mature peptide in human embryonic kidney 293 cells. Nedd4-2–mediated ubiquitination regulates lumenal relocation of SP-C, leading to processing and, ultimately, secretion of SP-C

Deficiency of SP-B reveals protective role of SP-C during oxygen lung injury

Although the surface properties of surfactant protein (SP)-B and SP-C are similar, the contributions that either protein may make to lung function have not been identified in vivo. Mutations in SP-B cause lethal respiratory failure at birth; however, SP-B null mice are deficient in both SP-B and SP-C. To identify potential contributions of SP-C to lung function in vivo, the following transgenic mice were generated and exposed to 95% O-2 for 3 days: (SP-B+/+,SP-C+/+), (SP-B+/+, SP-C-/-), (SP-B+/-,SP-C+/+), (SP-B+/-,SP-C+/+), and (SP-B+/-,SP-C-/-). Hyperoxia altered pressure-volume curves in mice that were heterozygous for SP- B, and these values were further decreased in (SP-B+/-,SP-C-/-) mice. Likewise, alveolar interleukin (IL)-6 and IL-1beta were maximally increased by O-2 exposure of (SP-B+/-,SP-C-/-) mice compared with the other genotypes. Lung hysteresivity was lower in the (SP-B+/-,SP-C-/-) mice. Surfactant isolated from (SP-B+/+,SP-C-/-) and (SP-B+/-,SP-C-/-) mice failed to stabilize the surface tension of microbubbles, showing that SP-C plays a role in stabilization or recruitment of phospholipid films at low bubble radius. Genetically decreased levels of SP-B combined with superimposed O-2-induced injury reveals the distinct contribution of SP-C to pulmonary function in vivo

The Benzenesulfoamide T0901317 [N-(2,2,2-Trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide] Is a Novel Retinoic Acid Receptor-Related Orphan Receptor-α/γ Inverse Agonist

Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we present the first high-affinity synthetic ligand for both RORα and RORγ. In a screen against all 48 human nuclear receptors, the benzenesulfonamide liver X receptor (LXR) agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) inhibited transactivation activity of RORα and RORγ but not RORβ. T0901317 was found to directly bind to RORα and RORγ with high affinity (Ki = 132 and 51 nM, respectively), resulting in the modulation of the receptor's ability to interact with transcriptional cofactor proteins. T0901317 repressed RORα/γ-dependent transactivation of ROR-responsive reporter genes and in HepG2 cells reduced recruitment of steroid receptor coactivator-2 by RORα at an endogenous ROR target gene (G6Pase). Using small interference RNA, we demonstrate that repression of the gluconeogenic enzyme glucose-6-phosphatase in HepG2 cells by T0901317 is ROR-dependent and is not due to the compound's LXR activity. In summary, T0901317 represents a novel chemical probe to examine RORα/γ function and an excellent starting point for the development of ROR selective modulators. More importantly, our results demonstrate that small molecules can be used to target the RORs for therapeutic intervention in metabolic and immune disorders

Targeting the adaptability of heterogeneous aneuploids

SummaryAneuploid genomes, characterized by unbalanced chromosome stoichiometry (karyotype), are associated with cancer malignancy and drug resistance of pathogenic fungi. The phenotypic diversity resulting from karyotypic diversity endows the cell population with superior adaptability. We show here, using a combination of experimental data and a general stochastic model, that the degree of phenotypic variation, thus evolvability, escalates with the degree of overall growth suppression. Such scaling likely explains the challenge of treating aneuploidy diseases with a single stress-inducing agent. Instead, we propose the design of an “evolutionary trap” (ET) targeting both karyotypic diversity and fitness. This strategy entails a selective condition “channeling” a karyotypically divergent population into one with a predominant and predictably drugable karyotypic feature. We provide a proof-of-principle case in budding yeast and demonstrate the potential efficacy of this strategy toward aneuploidy-based azole resistance in Candida albicans. By analyzing existing pharmacogenomics data, we propose the potential design of an ET against glioblastoma