Biochemical and structural studies of the interaction between ARNO and the Epidermal Growth Factor Receptor

Receptor tyrosine kinases (RTKs) and small GTP binding proteins (GTPases) are essential regulators of multiple cellular processes, making a tight control of their activity crucial for cellular homeostasis. Recently, it was shown that members of a class of guanine nucleotide exchange factors (GEFs), the cytohesin family, influence not only their canonical target proteins (small GTPases) but also the signaling of RTKs. Most prominently, they increase epidermal growth factor receptor (EGFR) activity by directly interacting with its intracellular domain. This mechanism is of pathophysiological relevance as demonstrated by in vivo studies in animal models and analysis of human tumor samples. In this study, I applied a combination of biochemical assays to further characterize the interaction between cytohesins and the EGFR. Using chemical crosslinking studies supplemented with microscale thermophoresis and fluorescence polarization experiments, I provide evidence for the direct interaction of the Sec7 domain of cytohesin 2 (ARNO) with the juxtamembrane (JM) domain of EGFR. Furthermore, the binding site was found to involve the C-terminus of ARNO Sec7 and the N-terminal region of EGFR JM. Together with functional data investigating the nucleotide exchange activity of ARNO, these results suggest a model in which the JM domain contacts the hydrophobic grove formed by helix F, G and H of ARNO Sec7. This interaction provides possible explanations for the positive regulation of EGFR kinase activity by ARNO. The discovery of non-canonical functions of cytohesins raises concerns about the use of GEF inhibitors like SecinH3 as specific inhibitors for Arf GTPases. Therefore, a high throughput screening (HTS) assay was established to identify small molecules that disrupt the interaction of active Arf6 with its specific effector protein JIP4. Due to very similar binding sites for different effector proteins on Arf6, molecules found in this assay are likely to exhibit a general inhibitory potential on GTPase signaling. The assay is based on fluorescence polarization (FP) and accurately tracks complex formation between Arf6 and the leucine zipper II (LZII) domain of JIP4. Tolerance towards detergents and organic solvents and an excellent Z’ value observed throughout the screening process indicate that the assay is suitable for future HTS applications using small molecule libraries optimized towards inhibitors for protein-protein-interactions

Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

<p>Abstract</p> <p>Background</p> <p>The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SR<it>β </it>subunit is an integral membrane protein, which tethers the SRP-interacting SR<it>α </it>subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SR<it>β </it>subunit and consists of only the SR<it>α </it>homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of <it>Escherichia coli</it>. So far, the exact function of the soluble SR in <it>E. coli </it>is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.</p> <p>Results</p> <p>In the current study we have determined the contribution of soluble FtsY to co-translational targeting <it>in vitro </it>and have re-analysed the localization of FtsY <it>in vivo </it>by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs) in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in <it>E. coli</it>. In agreement with this observation, our <it>in vivo </it>analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species <it>in vivo</it>.</p> <p>Conclusion</p> <p>The exact function of the SRP receptor (SR) in bacteria has so far been enigmatic. Our data show that the bacterial SR is almost exclusively membrane-bound <it>in vivo</it>, indicating that the presence of a soluble SR is probably an artefact of cell fractionation. Thus, co-translational targeting in bacteria does not involve the formation of a soluble SR-signal recognition particle (SRP)-ribosome nascent chain (RNC) intermediate but requires membrane contact of FtsY for efficient SRP-RNC recruitment.</p

Pharmacokinetic engineering of OX40-blocking anticalin proteins using monomeric plasma half-life extension domains

Anticalin® proteins have been proven as versatile clinical stage biotherapeutics. Due to their small size (∼20 kDa), they harbor a short intrinsic plasma half-life which can be extended, e.g., by fusion with IgG or Fc. However, for antagonism of co-immunostimulatory Tumor Necrosis Factor Receptor Superfamily (TNFRSF) members in therapy of autoimmune and inflammatory diseases, a monovalent, pharmacokinetically optimized Anticalin protein format that avoids receptor clustering and therefore potential activation is favored. We investigated the suitability of an affinity-improved streptococcal Albumin-Binding Domain (ABD) and the engineered Fab-selective Immunoglobulin-Binding Domain (IgBD) SpGC3Fab for plasma Half-Life Extension (HLE) of an OX40-specific Anticalin and bispecific Duocalin proteins, neutralizing OX40 and a second co-immunostimulatory TNFRSF member. The higher affinity of ABD fusion proteins to human serum albumin (HSA) and Mouse Serum Albumin (MSA), with a 4 to 5-order of magnitude lower KD compared with the binding affinity of IgBD fusions to human/mouse IgG, translated into longer terminal plasma half-lives (t1/2). Hence, the anti-OX40 Anticalin-ABD protein reached t1/2 values of ∼40 h in wild-type mice and 110 h in hSA/hFcRn double humanized mice, in contrast to ∼7 h observed for anti-OX40 Anticalin-IgBD in wild-type mice. The pharmacokinetics of an anti-OX40 Anticalin-Fc fusion protein was the longest in both models (t1/2 of 130 h and 146 h, respectively). Protein formats composed of two ABDs or IgBDs instead of one single HLE domain clearly showed longer presence in the circulation. Importantly, Anticalin-ABD and -IgBD fusions showed OX40 receptor binding and functional competition with OX40L-induced cellular reactivity in the presence of albumin or IgG, respectively. Our results suggest that fusion to ABD or IgBD can be a versatile platform to tune the plasma half-life of Anticalin proteins in response to therapeutic needs

Genome-Wide Phylogenetic Comparative Analysis of Plant Transcriptional Regulation: A Timeline of Loss, Gain, Expansion, and Correlation with Complexity

Evolutionary retention of duplicated genes encoding transcription-associated proteins (TAPs, comprising transcription factors and other transcriptional regulators) has been hypothesized to be positively correlated with increasing morphological complexity and paleopolyploidizations, especially within the plant kingdom. Here, we present the most comprehensive set of classification rules for TAPs and its application for genome-wide analyses of plants and algae. Using a dated species tree and phylogenetic comparative (PC) analyses, we define the timeline of TAP loss, gain, and expansion among Viridiplantae and find that two major bursts of gain/expansion occurred, coinciding with the water-to-land transition and the radiation of flowering plants. For the first time, we provide PC proof for the long-standing hypothesis that TAPs are major driving forces behind the evolution of morphological complexity, the latter in Plantae being shaped significantly by polyploidization and subsequent biased paleolog retention. Principal component analysis incorporating the number of TAPs per genome provides an alternate and significant proxy for complexity, ideally suited for PC genomics. Our work lays the ground for further interrogation of the shaping of gene regulatory networks underlying the evolution of organism complexity

The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3'UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3' UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3' UTR

The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3′ UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3′ UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3′ UTR

High concentration formulation developability approaches and considerations

ABSTRACTThe growing need for biologics to be administered subcutaneously and ocularly, coupled with certain indications requiring high doses, has resulted in an increase in drug substance (DS) and drug product (DP) protein concentrations. With this increase, more emphasis must be placed on identifying critical physico-chemical liabilities during drug development, including protein aggregation, precipitation, opalescence, particle formation, and high viscosity. Depending on the molecule, liabilities, and administration route, different formulation strategies can be used to overcome these challenges. However, due to the high material requirements, identifying optimal conditions can be slow, costly, and often prevent therapeutics from moving rapidly into the clinic/market. In order to accelerate and derisk development, new experimental and in-silico methods have emerged that can predict high concentration liabilities. Here, we review the challenges in developing high concentration formulations, the advances that have been made in establishing low mass and high-throughput predictive analytics, and advances in in-silico tools and algorithms aimed at identifying risks and understanding high concentration protein behavior

Depletion of the Signal Recognition Particle Receptor Inactivates Ribosomes in Escherichia coli▿

The signal recognition particle (SRP)-dependent cotranslational targeting of proteins to the cytoplasmic membrane in bacteria or the endoplasmic reticulum membrane in eukaryotes is an essential process in most living organisms. Eukaryotic cells have been shown to respond to an impairment of the SRP pathway by (i) repressing ribosome biogenesis, resulting in decreased protein synthesis, and (ii) by increasing the expression of protein quality control mechanisms, such as chaperones and proteases. In the current study, we have analyzed how bacteria like Escherichia coli respond to a gradual depletion of FtsY, the bacterial SRP receptor. Our analyses using cell-free transcription/translation systems showed that FtsY depletion inhibits the translation of both SRP-dependent and SRP-independent proteins. This synthesis defect is the result of a multifaceted response that includes the upregulation of the ribosome-inactivating protein ribosome modulation factor (RMF). Although the consequences of these responses in E. coli are very similar to some of the effects also observed in eukaryotic cells, one striking difference is that E. coli obviously does not reduce the rate of protein synthesis by downregulating ribosome biogenesis. Instead, the upregulation of RMF leads to a direct and reversible inhibition of translation