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.

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.

Characterization of a human pluripotent stem cell-derived model of neuronal development using multiplexed targeted proteomics.

Purpose: Human pluripotent stem cell (hPSC)-derived cellular models have great potential to enable drug discovery and improve translation of preclinical insights to the clinic. We have developed a hPSC-derived neural precursor cell model for studying early events in human brain development. We present protein-level characterization of this model, using a multiplexed SRM approach, to establish reproducibility and physiological relevance; essential prerequisites for utilization of the neuronal development model in phenotypic screening-based drug discovery.

Quantitative ADME proteomics - CYP and UGT enzymes in the Beagle dog liver and intestine.

Purpose: Beagle 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.

Methods: Abundances of enzymes were measured in Beagle dog intestine and liver using selected reaction monitoring mass spectrometry.

Determination of free desmosine in human plasma and its application in two experimental medicine studies.

Elastin is one of the major extracellular matrix proteins associated with connective tissue. Its degradation leads to the liberation of the unique amino acids desmosine and isodesmosine. These have shown utility as biomarkers of elastin breakdown for disease progression, patient stratification, and drug efficacy.

Mass spectrometry-based quantification of CYP enzymes to establish in vitro/in vivo scaling factors for intestinal and hepatic metabolism in beagle dog.

Purpose: Physiologically based models, when verified in pre-clinical species, optimally predict human pharmacokinetics. However, modeling of intestinal metabolism has been a gap. To establish in vitro/in vivo scaling factors for metabolism, the expression and activity of CYP enzymes were characterized in the intestine and liver of beagle dog.

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.

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.

Direct interactions between subunits of CPSF and the U2 snRNP contribute to the coupling of pre-mRNA 3' end processing and splicing.

Eukaryotic pre-mRNAs are capped at their 5' ends, polyadenylated at their 3' ends, and spliced before being exported from the nucleus to the cytoplasm. Although the three processing reactions can be studied separately in vitro, they are coupled in vivo. We identified subunits of the U2 snRNP in highly purified CPSF and showed that the two complexes physically interact.

Pro-invasive gene regulating effect of irradiation and combined temozolomide-radiation treatment on surviving human malignant glioma cells.

The current chemotherapeutic treatment of glioblastoma patients has minor success. Little is known about the molecular and cellular mechanisms of the resistance of gliomas towards current therapies. This study investigated both suppressive cellular effects and regulation of extracellular matrix remodeling proteins with pro-invasive activity in surviving human glioblastoma cells under clinically relevant treatments.

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.

Is current therapy of malignant gliomas beneficial for patients? Proteomics evidence of shifts in glioma cells expression patterns under clinically relevant treatment conditions.

The most common human brain tumours - gliomas - have poor prognosis with and without treatment. The current therapy conditions act sub-lethally and cannot effectively suppress the proliferation of glioma cells. Here we show differential protein expression patterns in surviving human malignant U87-MG glioma cells under clinically relevant chemo/radiotherapy.

A 13 kDa carboxy-terminal fragment of ApoE stabilizes Abeta hexamers.

The pathological role of ApoE4 in Alzheimer's disease (AD) is not fully elucidated yet but there is strong evidence that ApoE is involved in Abeta deposition, which is an early hallmark of AD neuropathology. Overexpression of ApoE in neuroblastoma cells (Neuro2a) leads to the generation of an intracellular 13 kDa carboxy-terminal fragment of ApoE comparable to fragments seen in brains of AD patients. ApoE4 generates more of this fragment than ApoE2 and E3 suggesting a potential pathological role of these fragments in Alzheimer's disease.

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 (tRNAiMet). 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.

Parkin phosphorylation and modulation of its E3 ubiquitin ligase activity.

Mutations in the PARKIN gene are the most common cause of hereditary parkinsonism. The parkin protein comprises an N-terminal ubiquitin-like domain, a linker region containing caspase cleavage sites, a unique domain in the central portion, and a special zinc finger configuration termed RING-IBR-RING. Parkin has E3 ubiquitin-protein ligase activity and is believed to mediate proteasomal degradation of aggregation-prone proteins.

The serum proteome of Equus caballus.

We constructed a reference two-dimensional protein map for horse (Equus caballus) serum. The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified. Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology.

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.

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

The Alzheimer's disease-associated presenilin (PS) 1 is intimately involved in gamma-secretase cleavage of beta-amyloid precursor protein and other proteins. In addition, PS1 plays a role in beta-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.

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.

A role for SSU72 in balancing RNA polymerase II transcription elongation and termination.

Interactions of pre-mRNA 3'end factors and the CTD of RNA polymerase II (RNAP II) are required for transcription termination and 3'end processing. Here, we demonstrate that Ssu72p is stably associated with yeast cleavage and polyadenylation factor CPF and provide evidence that it bridges the CPF subunits Pta1p and Ydh1p/Cft2p, the general transcription factor TFIIB, and RNAP II via Rpb2p. Analyses of ssu72-2 mutant cells in the absence and presence of the nuclear exosome component Rrp6p revealed defects in RNAP II transcription elongation and termination.

Changes in the levels of low-abundance brain proteins induced by kainic acid.

Low-abundance gene products are of interest in proteomic studies, because they are probably involved in disease-related changes and their altered levels or modifications may carry significant biological information. Detection of low-abundance proteins of a proteome is one of the major limitations of proteomics and a scientific challenge. We investigated the changes in the levels of low-abundance rat brain cytosolic proteins after administration of kainic acid, a potent neurotoxin and excitatory amino acid.

Expression pattern and localization of beta,beta-carotene 15,15'-dioxygenase in different tissues.

Beta,beta-carotene 15,15'-dioxygenase cleaves beta,beta-carotene into two molecules of retinal, and is the key enzyme in the metabolism of beta,beta-carotene to vitamin A. The enzyme has been known for more than 40 years, yet all attempts to purify the protein to homogeneity have failed. Recently, the successful cloning and sequencing of an enzyme with beta,beta-carotene 15,15'-dioxygenase activity from chicken, as well as from Drosophila, has been reported.

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.

Cloning and expression of beta,beta-carotene 15,15'-dioxygenase.

beta,beta-Carotene 15,15'-dioxygenase cleaves beta-carotene into two molecules of retinal and is therefore the key enzyme in beta-carotene metabolism to vitamin A. In the present study, it was possible to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent that partial amino acid sequence information could be obtained to design degenerate oligonucleotides. With RT-PCR a cDNA fragment could be obtained and used subsequently in a radioactive screening of a chicken duodenal expression library.

Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells.

Angiotensinogen, the renin (E.C. 3.