Pay32p of the Yeast Yarrowia lipolytica Is an Intraperoxisomal Component of the Matrix Protein Translocation Machinery

Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay32-1, has abnormally small peroxisomes that are often found in clusters surrounded by membranous material. The functionally complementing gene PAY32 encodes a protein, Pay32p, of 598 amino acids (66,733 D) that is a member of the tetratricopeptide repeat family. Pay32p is intraperoxisomal. In wild-type peroxisomes, Pay32p is associated primarily with the inner surface of the peroxisomal membrane, but ~30% of Pay32p is localized to the peroxisomal matrix. The majority of Pay32p in the matrix is complexed with two polypeptides of 62 and 64 kD recognized by antibodies to SKL (peroxisomal targeting signal-1). In contrast, in peroxisomes of the pay32-1 mutant, Pay32p is localized exclusively to the matrix and forms no complex. Biochemical characterization of the mutants pay32-1 and pay32-KO (a PAY32 gene disruption strain) showed that Pay32p is a component of the peroxisomal translocation machinery. Mutations in the PAY32 gene prevent the translocation of most peroxisome-bound proteins into the peroxisomal matrix. These proteins, including the 62-kD anti-SKL-reactive polypeptide, are trapped in the peroxisomal membrane at an intermediate stage of translocation in pay32 mutants. Our results suggest that there are at least two distinct translocation machineries involved in the import of proteins into peroxisomes.

Neto1 Is a Novel CUB-Domain NMDA Receptor–Interacting Protein Required for Synaptic Plasticity and Learning

The N-methyl-D-aspartate receptor (NMDAR), a major excitatory ligand-gated ion channel in the central nervous system (CNS), is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1), a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP) at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans

The CIP2A–TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers

The MMS22L-TONSL Complex Mediates Recovery from Replication Stress and Homologous Recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Control of late off-center cone bipolar cell differentiation and visual signaling by the homeobox gene Vsx1.

Retinal bipolar cells are interneurons that transmit visual signals from photoreceptors to ganglion cells. Although the visual pathways mediated by bipolar cells have been well characterized, the genes that regulate their development and function are largely unknown. To determine the role in bipolar cell development of the homeobox gene Vsx1, whose retinal expression is restricted to a major subset of differentiating and mature cone bipolar (CB) cells, we targeted the gene in mice. Bipolar cell fate was not altered in the absence of Vsx1 function, because the pan-bipolar markers Chx10 and Ret-B1 continued to be expressed in inner nuclear layer neurons labeled by the Vsx1-targeting reporter gene, tauLacZ. The specification, number, and gross morphology of the subset of on-center and off-center (OFF)-CB cells defined by tauLacZ expression from the Vsx1 locus were also normal in Vsx1(tauLacZ)/Vsx1(tauLacZ) mice. However, the terminal differentiation of OFF-CB cells in the retina of Vsx1(tauLacZ)/Vsx1(tauLacZ) mice was incomplete, as demonstrated by a substantial reduction in the expression of at least four markers (recoverin, NK3R, Neto1, and CaB5) for these interneurons. These molecular abnormalities were associated with defects in retinal function and documented by electroretinography and in vitro ganglion cell recordings specific to cone visual signaling. In particular, there was a general reduction in the light-mediated activity of OFF, but not on-center, ganglion cells. Thus, Vsx1 is required for the late differentiation and function of OFF-CB cells and is associated with a heritable OFF visual pathway-specific retinal defect

The PAH2 gene is required for peroxisome assembly in the methylotrophic yeast Hansenula polymorpha and encodes a member of the tetratricopeptide repeat family of proteins

Peroxisome assembly mutants in the methylotrophic yeast, Hansenula polymorpha, were selected by a novel procedure involving the inability of mutants to use both oleic acid and methanol as carbon sources. These compounds are both metabolized within peroxisomes through two different enzymatic pathways. 15 mutant strains called mut (methanol non-utilizing) were isolated. These strains were assigned to ten genetic complementation groups. Subcellular fractionation analysis showed that peroxisomal matrix enzymes were mislocalized to the cytoplasm in mut strains. Electron microscopy confirmed that the inability of mut strains to grow on oleic acid and methanol was due to defects in peroxisome assembly. Functional complementation of a mutant strain, mut2, with a plasmid library of H. polymorpha genomic DNA sequences has identified a gene, PAH2, that restores growth on methanol and the correct localization of matrix enzymes to the peroxisome. PAH2 encodes Pah2p, a polypeptide of 569 amino acids that is a member of the tetratricopetide repeat (TPR) family of proteins. Pah2p shows identity with Pas8p and Pas10p, two proteins required for peroxisome assembly in the yeasts Pichia pastoris and Saccharomyces cerevisiae, respectively, and which have been suggested to be receptors that recognize peroxisomal targeting signal-1 (PTS1) motifs.

The Yarrowia lipolytica Gene PAY5 Encodes a Peroxisomal Integral Membrane Protein Homologous to the Mammalian Peroxisome Assembly Factor PAF-1

Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay5-1, lacks normal peroxisomes and instead contains irregular vesicular structures surrounded by multiple unit membranes. The pay5-1 mutant is not totally deficient in peroxisomal matrix protein targeting, as a subset of matrix proteins continues to localize to a subcellular fraction enriched for peroxisomes. The functionally complementing gene PAY5 encodes a protein, Pay5p, of 380 amino acids (41,720 Da). Pay5p is a peroxisomal integral membrane protein homologous to mammalian PAF-1 proteins, which are essential for peroxisome assembly and whose mutation in humans results in Zellweger syndrome. Pay5p is targeted to mammalian peroxisomes, demonstrating the evolutionary conservation of the targeting mechanism for peroxisomal membrane proteins. Our results suggest that in pay5 mutants, normal peroxisome assembly is blocked, which leads to the accumulation of the membranous vesicular structures observed.