The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore.

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore

A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA(Met) (tRNA(i) (Met)). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNA(i) (Met) with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNA(i) (Met) by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover

Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors

Six different protein factors are required in vitro for 3′ end formation of mammalian pre-mRNAs by endonucleolytic cleavage and polyadenylation. Five of the factors have been purified and most of their components cloned, but cleavage factor II(m) (CF II(m)) remained uncharacterized. We have purified CF II(m) from HeLa cell nuclear extract by several chromatographic steps. During purification, CF II(m) activity separated into two components, one essential (CF IIA(m)) and one stimulatory (CF IIB(m)) for the cleavage reaction. CF IIA(m) fractions contain the human homologs of two yeast 3′ end processing factors, Pcf11p and Clp1p, as well as cleavage factor I(m) (CF I(m)) and several splicing and transcription factors. We report the cloning of hClp1 and show that it is a genuine subunit of CF IIA(m). Antibodies directed against hClp1 deplete cleavage activity, but not polyadenylation activity from HeLa cell nuclear extract. hClp1 interacts with CF I(m) and the cleavage and polyadenylation specificity factor CPSF, suggesting that it bridges these two 3′ end processing factors within the cleavage complex

Use of the Immunodominant 18-Kilodalton Small Heat Shock Protein as a Serological Marker for Exposure to Mycobacterium ulcerans

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease

mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2+/− and TSC2−/− neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis

Matrix Biology / N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier. For quantitative secretome analysis, project accession: PXD007691, username: [email protected], password: 1FUpS6Yq. For TAILS analysis, project accession: PXD007139, username: [email protected], password: sNbMp7xK.(VLID)360006

Trf4p Is the Catalytic Subunit of a New Poly(A) Polymerase

<div><p>(A) The Trf4 complex has poly(A) polymerase activity. The 5′-end-labeled oligo(A)₁₅ was incubated 30 min with 5, 10, or 20 ng of affinity-purified fractions of the wild-type TAP-tagged Trf4p (Trf4-TAP) or mutant Trf4p with the aspartic acid residues 236 and 238 changed to alanines (DADA-TAP). Protein was omitted in lane 1. Recombinant yeast poly(A) polymerase (Pap1), 1, 2, and 4 ng, was used as a positive control. The migration position of oligo(A)₁₅ is indicated by an arrow.</p> <p>(B) The Trf4p activity is specific for the addition of adenosine monophosphate. Polyadenylation assays with 20 ng of Trf4-TAP in the presence of different ribonucleoside triphosphates. Recombinant yeast Pap1p, 5 ng, was used as a control. All samples were separated on 15% denaturing gels.</p></div

The Polyadenylation Activity of the Trf4 Complex Stimulates the Degradation of Unmodified tRNA_i^Met by the Nuclear Exosome

<div><p>(A) The PAP activity of Trf4 complex is required to stimulate the exosome activity. In a coupled exosome/polyadenylation assay, 5′-end-labeled unmodified tRNA_i^Met was incubated with 50 ng of affinity-purified Rrp6-TAP eluate for 30 min as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030189#s4" target="_blank">Materials and Methods</a> (lane 2), followed by addition of 50 ng of wild-type (Trf4-TAP), mutant complex (DADA-TAP), or buffer A (buffer). Reactions were stopped after 10 (lanes 3, 7, and 11), 30 (lanes 4, 8, and 12), 60 (lanes 5, 9, and 13), or 90 min (lanes 6, 10, and 14) and separated on a 15% gel. Arrows indicate the position of the input tRNA. Protein was omitted in lane 1 of each gel. The migration positions of the degradation products (dp) are indicated by a bracket.</p> <p>(B) Coupled polyadenylation/exosome assay. The 5′-end-labeled unmodified tRNA_i^Met was pre-adenylated with 50 ng of affinity-purified Trf4-TAP complex for 30 min (lane 2). Then 50 ng of exosome complex (Rrp6-TAP) or buffer A (buffer) was added, and the reactions were continued as in (A).</p> <p>(C) Depletion of Mtr4p results in incomplete degradation. Coupled-assay, 5′-end-labeled unmodified tRNA_i^Met was pre-incubated for 30 min with Trf4p-TAP lacking Mtr4p (Trf4-TAP w/o Mtr4), followed by the addition of 50 ng of Rrp6-TAP complex or buffer A, and the incubation was continued as in (A).</p></div