Johannes Grillari - Academia.edu (original) (raw)

Papers by Johannes Grillari

New biotechnology, Jan 18, 2015

Transient gene expression (TGE) is an essential tool for the production of recombinant proteins, ... more Transient gene expression (TGE) is an essential tool for the production of recombinant proteins, especially in early drug discovery and development phases of biopharmaceuticals. The need for fast production of sufficient recombinant protein for initial tests has dramatically increased with increase in the identification of potential novel pharmaceutical targets. One of the critical factors for transient transfection is plasmid copy number (PCN), for which we here provide an optimized qPCR based protocol. Thereby, we show the loss of PCN during a typical batch process of HEK293 cells after transfection from 606,000 to 4560 copies per cell within 5 days. Finally two novel human kidney cell lines, RS and RPTEC/TERT1 were compared to HEK293 and proved competitive in terms of PCN and specific productivity. In conclusion, since trafficking and degradation of plasmid DNA is not fully understood yet, improved methods for analysis of PCN may contribute to design specific and more stable plas...

New biotechnology, Jan 18, 2014

The market of recombinant proteins as human pharmaceuticals has surpassed annual revenues of more... more The market of recombinant proteins as human pharmaceuticals has surpassed annual revenues of more than 150 billion dollars. The marketed proteins are often complex in terms of post-translational modifications and conventional hosts have shown weaknesses in terms of quality of these recombinant proteins. Especially the non-human glycopatterns leading to immunogenicity or shortened in vivo half-life have gained attention over the past decade. Therefore, production cell lines with better or novel characteristics are required and human cell lines seem to be the most genuine and logical choice. Thus, several human cell lines have been used to generate biopharmaceuticals. We here present an overview of such examples and highlight their promise for biopharmaceutical production processes of the future.

Aging, 2012

Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutation... more Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutations in DNA repair factors lead to accelerated and premature aging. However, the opposite, namely if improved DNA repair will also increase the life or health span is less clear, and only few studies have tested if overexpression of DNA repair factors modulates life and health span in cells or organisms. Recently, we identified and characterized SNEVhPrp19/hPso4, a protein that plays a role in DNA repair and pre-mRNA splicing, and observed a doubling of the replicative life span upon ectopic overexpression, accompanied by lower basal DNA damage and apoptosis levels as well as an increased resistance to oxidative stress. Here we find that SNEVhPrp19/hPso4 is phosphorylated at S149 in an ataxia telangiectasia mutated protein (ATM)-dependent manner in response to oxidative stress and DNA double strand break inducing agents. By overexpressing wild-type SNEVhPrp19/hPso4 and a phosphorylation-def...

Biotechnology and Bioengineering, 2015

MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of ... more MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from the Chinese Hamster (CGR) and Chinese hamster ovary (CHO) cells provide new tools for the discovery of novel miRNAs in this important production system. Version 20 of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences for CGR/CHO. We searched for evolutionary conserved miRNAs from miRBase v20 in recently published genomic data, derived from Chinese hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several hundred miRNA sequences in the genome. For several of these, the expression in CHO cells could be verified from multiple next-generation sequencing experiments. In addition, several hundred unexpressed miRNAs are awaiting further confirmation by testing for their transcription in different Chinese hamster tissues. This article is protected by copyright. All rights reserved.

Protein Metabolism and Homeostasis in Aging, 2010

Ubiquitination ofendogenous proteins is one of the key regulatory steps that guides protein degra... more Ubiquitination ofendogenous proteins is one of the key regulatory steps that guides protein degradation through regulation of proteasome activity. During the last years evidence has accumulated that proteasome activity is decreased during the aging process in various model systems and that these changes might be causally related to aging and age-associated diseases. Since in most instances ubiquitination is the primary event in target selection, the system ofubiquitination and deubiquitination might be of similar importance. Furthermore, ubiquitination and proteasomal degradation are not completely congruent, since ubiquitination confers also functions different from targeting proteins for degradation. Depending on mono- and polyubiquitination and on how ubiquitin chains are linked together, post-translational modifications of cellular proteins by covalent attachment of ubiquitin and ubiquitin-like proteins are involved in transcriptional regulation, receptor internalization, DNA repair, stabilization of protein complexes and autophagy. Here, we summarize the current knowledge regarding the ubiquitinome and the underlying ubiquitin ligases and deubiquitinating enzymes in replicative senescence, tissue aging as well as in segmental progeroid syndromes and discuss potential causes and consequences for aging.

Mesenchymal Stem Cells - Basics and Clinical Application I, 2012

Traditionally, clinicians and researchers have relied on a skin biopsy or blood extraction as rel... more Traditionally, clinicians and researchers have relied on a skin biopsy or blood extraction as relatively accessible supplies for in vitro cell expansion and biological studies. Perhaps surprisingly, limited attention has been given to a totally noninvasive source, urine, which eliminates the discomfort associated with other procedures. This may arise from the perception that urine is merely a body waste. Yet, the analysis of urine is a longstanding fundamental test for diagnostic purposes and nowadays there is growing interest in using urine for detecting biomarkers. In addition, recent work including ours reinforces the idea that urine contains a variety of viable cell types with relevant applications. In this review, we describe those cell types and their potential uses.

New Biotechnology, 2014

Background: Oct4 is a transcription factor that plays a major role for the preservation of the pl... more Background: Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells.

BMC Proceedings, 2011

Since 1957 Chinese hamster ovary (CHO) cells are used for in vitro cultivation as they require as... more Since 1957 Chinese hamster ovary (CHO) cells are used for in vitro cultivation as they require assimilable low sustenance . Today, CHO cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins and are considered as the mammalian equivalent of E. coli in research and biotechnology . The production of biopharmaceuticals in CHO cells is superior to protein production in bacteria, because mammalian cell lines procure complex folding and post-translational modifications like glycosylation. However, contrary to the increasing importance in biotechnology and industry, comprehensive genome and transcriptome information of CHO cell lines is still rare.

Nature Communications, 2015

Several pathways modulating longevity and stress resistance converge on translation by targeting ... more Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Scientific Reports, 2014

Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms respons... more Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.

Applied Microbiology and Biotechnology, 2014

MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene express... more MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and nonproducing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product-and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of highthroughput microRNA expression data in CHO cells.

Tissue Engineering Part A, 2009

Cell banking of mesenchymal stem cells (SCs) from various human tissues has significantly increas... more Cell banking of mesenchymal stem cells (SCs) from various human tissues has significantly increased the feasibility of SC-based therapies. Sources such as adipose tissue and amnion offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scaleable production and long-term analysis of these cells. Here we report the establishment and characterization of two human amnion-derived and two human adipose-derived SC lines immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT). hTERT overexpression resulted in continuously growing SC lines that were largely unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differentiation potential for up to 87 population doublings. While all generated lines showed equal immunomodulation compared to the parental cells, one of the amnion-derived immortalized lines resulted in significantly increased immunogenicity. Although telomerase proves as important tool for immortalizing cells, our data emphasize the need for careful and standardized characterization of each individual cell population for cell banks.

PLoS ONE, 2012

Originally the novel protein Blom7a was identified as novel pre-mRNA splicing factor that interac... more Originally the novel protein Blom7a was identified as novel pre-mRNA splicing factor that interacts with SNEV Prp19/Pso4 , an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7a belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A) 1-4 C 2-6 (U/A) 1-5 , we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7a with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7a might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development.

Oncogene, 2012

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene underlies the majority of spora... more Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene underlies the majority of sporadic clear cell renal cell carcinomas (CCRCCs) and is also responsible for the hereditary VHL cancer syndrome. VHL loss of function results in constitutive stabilization of hypoxia-inducible factors (HIF-1a and HIF-2a) due to insufficient proteolysis in the presence of oxygen. This activates multiple genes relevant to tumorigenesis, allowing cells to acquire further mutations and undergo malignant transformation. However, the specific role of each HIF-a subunit in CCRCC tumorigenesis is not yet well understood. The current paradigm supports that in the first stages of CCRCC formation the stabilization of HIF-1a is dominant and this limits proliferation, but later on HIF-2a increases and this induces a more aggressive cell behavior. Understanding how this transition happens is highly relevant, as it may provide novel ways to treat these cancers. Here, we show that VHL inactivation in CCRCC cells results in HIF-1a/2a-dependent downregulation of HIF-1a mRNA through direct binding of either subunit to a reverse hypoxia-response element in the HIF-1a proximal promoter. This binding activates a series of repressive histone modification marks including histone 3 lysine 27 trimethylation (H3K27me3) to make the changes stable, and if overturned reduces CCRCC cell proliferation due to excessive HIF-1a expression level. Our findings thus help understand how HIF-a subunits influence each other and also reinforce the idea that epigenetic mechanisms are a key step of CCRCC progression.

Nucleic Acids Research, 2005

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequen... more We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A humanyeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein-protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56-74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homooligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.