Arno Friedlein

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 tRNAMet (tRNAi 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 tRNAi 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 tRNAi 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.

Activating interactions in human NK cell recognition: the role of 2B4‐CD48

2B4 is a cell surface glycoprotein of the immunoglobulin superfamily structurally related to CD2‐like molecules. It was originally identified in the mouse as a receptor that mediates non‐MHC‐restricted cytotoxicity by NK cells and CD8+ T cells. Recently, 2B4 was shown to bind CD48 by molecular binding assays and surface plasmon resonance. Here, we have investigated the cell surface expression, biochemical characteristics and function of human 2B4. Our results show that 2B4 is expressed not only on NK cells and CD8+ T cells, but also on monocytes and basophils, indicating a broader role for 2B4 in leukocyte activation. In NK cells, engagement of 2B4 with a specific monoclonal antibody or with CD48 can trigger NK cell‐mediated cytotoxicity. The contribution of 2B4‐CD48 interaction to target cell lysis by different NK cell clones varies, probably dependent on the relative contribution of other receptor‐ligand interactions. In T cells and monocytes, ligation of 2B4 does not lead to T cell or monocyte activation. Thus, it appears that the primary function of 2B4 is to modulate other receptor‐ligand interactions to enhance leukocyte activation.

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

In mammals, polyadenylation of mRNA precursors (pre‐mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U‐rich upstream sequence elements on the pre‐mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine‐rich RNA‐binding motif that preferentially binds to U‐rich sequence elements on the pre‐mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U‐rich element‐dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF‐mediated stimulation of PAP activity.

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 IIm (CF IIm) remained uncharacterized. We have purified CF IIm from HeLa cell nuclear extract by several chromatographic steps. During purification, CF IIm activity separated into two components, one essential (CF IIAm) and one stimulatory (CF IIBm) for the cleavage reaction. CF IIAm fractions contain the human homologs of two yeast 3′ end processing factors, Pcf11p and Clp1p, as well as cleavage factor Im (CF Im) and several splicing and transcription factors. We report the cloning of hClp1 and show that it is a genuine subunit of CF IIAm. Antibodies directed against hClp1 deplete cleavage activity, but not polyadenylation activity from HeLa cell nuclear extract. hClp1 interacts with CF Im and the cleavage and polyadenylation specificity factor CPSF, suggesting that it bridges these two 3′ end processing factors within the cleavage complex.

Biomarker Discovery by Imaging Mass Spectrometry Transthyretin is a Biomarker for Gentamicin-induced Nephrotoxicity in Rat

Adverse drug effects are often associated with pathological changes in tissue. An accurate depiction of the undesired affected area, possibly supported by mechanistic data, is important to classify the effects with regard to relevance for human patients. MALDI imaging MS represents a new analytical tool to directly provide the spatial distribution and the relative abundance of proteins in tissue. Here we evaluate this technique to investigate potential toxicity biomarkers in kidneys of rats that were administered gentamicin, a well known nephrotoxicant. Differential analysis of the mass spectrum profiles revealed a spectral feature at 12,959 Da that strongly correlates with histopathology alterations of the kidney. We unambiguously identified this spectral feature as transthyretin (Ser28–Gln146) using an innovative combination of tissue microextraction and fractionation by reverse-phase liquid chromatography followed by a top-down tandem mass spectrometric approach. Our findings clearly demonstrate the emerging role of imaging MS in the discovery of toxicity biomarkers and in obtaining mechanistic insights concerning toxicity mechanisms.

Phosphorylation of presenilin 1 at the caspase recognition site regulates its proteolytic processing and the progression of apoptosis

The Alzheimers disease-associated presenilin (PS) 1 is intimately involved in γ-secretase cleavage of β-amyloid precursor protein and other proteins. In addition, PS1 plays a role in β-catenin signaling and in the regulation of apoptosis. Here we demonstrate that phosphorylation of PS1 is regulated by two independent signaling pathways involving protein kinase (PK) A and PKC and that both kinases can directly phosphorylate the large hydrophilic domain of PS1 in vitro and in cultured cells. A phosphorylation site at serine residue 346 was identified that is selectively phosphorylated by PKC but not by PKA. This site is localized within a recognition motif for caspases, and phosphorylation strongly inhibits proteolytic processing of PS1 by caspase activity during apoptosis. Moreover, PS1 phosphorylation reduces the progression of apoptosis. Our data indicate that phosphorylation/dephosphorylation at the caspase recognition site provides a mechanism to reversibly regulate properties of PS1 in apoptosis.

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

ABSTRACT 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.

Hydrophilic interaction chromatography of intact, soluble proteins.

The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.

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.

Quantitative ADME Proteomics – CYP and UGT Enzymes in the Beagle Dog Liver and Intestine

PurposeBeagle dogs are used to study oral pharmacokinetics and guide development of drug formulations for human use. Since mechanistic insight into species differences is needed to translate findings in this species to human, abundances of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) drug metabolizing enzymes have been quantified in dog liver and intestine.MethodsAbundances of enzymes were measured in Beagle dog intestine and liver using selected reaction monitoring mass spectrometry.ResultsSeven and two CYPs were present in the liver and intestine, respectively. CYP3A12 was the most abundant CYP in both tissues. Seven UGT enzymes were quantified in the liver and seven in the intestine although UGT1A11 and UGT1A9 were present only in the intestine and UGT1A7 and UGT2B31 were found only in the liver. UGT1A11 and UGT1A2 were the most abundant UGTs in the intestine and UGT2B31 was the most abundant UGT in the liver. Summed abundance of UGT enzymes was similar to the sum of CYP enzymes in the liver whereas intestinal UGTs were up to four times more abundant than CYPs. The estimated coefficients of variation of abundance estimates in the livers of 14 donors were separated into biological and technical components which ranged from 14 to 49% and 20 to 39%, respectively.ConclusionsAbundances of canine CYP enzymes in liver and intestine have been confirmed in a larger number of dogs and UGT abundances have been quantified for the first time. The biological variability in hepatic CYPs and UGTs has also been estimated.