[Show abstract][Hide abstract] ABSTRACT: The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the β subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the β subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell - free system. Vps4A was validated for direct binding to the FTase α subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase α controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase α subunit that includes control of intracellular membrane trafficking.
No preview · Article · Nov 2015 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Electrochemical biosensors convert biological events to an electrical current. To date most electrochemical biosensors exploit activities of naturally occurring enzymes. Here we demonstrated that insertion of a calmodulin domain into the redox enzyme PQQ-glucose dehydrogenase resulted in a selective Ca(2+) biosensor that could be used to rapidly measure Ca(2+) concentrations in human biological fluids. We were able to convert a point-of-care glucometer into Ca(2+) monitor by refurbishing it with the developed biosensor. We propose that similar engineering strategies may be used to create highly specific electrochemical biosensors to other analytes. Compatibility with cheap and ubiquitous amperometric detectors is expected to accelerate progression of these biosensors into clinical applications.
No preview · Article · Nov 2015 · Chemical Communications
[Show abstract][Hide abstract] ABSTRACT: Bisphosphonate drugs such as zoledronic acid (ZOL), used for the treatment of common bone disorders, target the skeleton and inhibit bone resorption by preventing the prenylation of small GTPases in bone-destroying osteoclasts. Increasing evidence indicates that bisphosphonates also have pleiotropic effects outside the skeleton, most likely via cells of the monocyte/macrophage lineage exposed to nanomolar circulating drug concentrations. However, no effects of such low concentrations of ZOL have been reported using existing approaches. We have optimised a highly sensitive in vitro prenylation assay utilising recombinant geranylgeranyltransferases to enable the detection of subtle effects of ZOL on the prenylation of Rab- and Rho-family GTPases. Using this assay, we found for the first time that concentrations of ZOL as low as 10 nM caused inhibition of Rab prenylation in J774 macrophages following prolonged cell culture. By combining the assay with quantitative mass spectrometry we identified an accumulation of 18 different unprenylated Rab proteins in J774 cells after nanomolar ZOL treatment, with a >7-fold increase in the unprenylated form of Rab proteins associated with the endophagosome pathway (Rab1, Rab5, Rab6, Rab7, Rab11, Rab14 and Rab21). Finally, we also detected a clear effect of subcutaneous ZOL administration in vivo on the prenylation of Rab1A, Rab5B, Rab7A and Rab14 in mouse peritoneal macrophages, confirming that systemic treatment with bisphosphonate drug can inhibit prenylation in myeloid cells in vivo outside the skeleton. These observations begin a new era in defining the precise pharmacological actions of bisphosphonate drugs on the prenylation of small GTPases in vivo.
[Show abstract][Hide abstract] ABSTRACT: Over the last half a century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms havehas been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the Species- Independent Translation Initiation sequence to express and characterise 88 N-terminallyterminaly GFP- tagged human cytosolic proteins of different sizes in E. coli, wheat germWheat Germ (WGE),), HeLa and Leishmania-based (LTE) cell-free systems. Using a combination of single- molecule fluorescence spectroscopy, SDS-PAGE and Western blot analysis we assessedas assess the expression yields, the fraction of full- length translation product and its aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species— particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systemssystem tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, while the LTE- produced proteins have the lowest aggregation propensity among the systems analysed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. This article is protected by copyright. All rights reserved
No preview · Article · Aug 2015 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.
No preview · Article · Mar 2015 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in Saccharomy-ces cerevisiae based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in S. cerevisiae. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltrans-ferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery.
[Show abstract][Hide abstract] ABSTRACT: The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.
Preview · Article · Oct 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impacts on neuroexocytosis in this pathology is unknown. Using neurosecretory cells deficient in Munc18, we show that a disease-linked mutation, C180Y, renders the protein unstable at 37°C. Although the mutated protein retains its function as t-SNARE chaperone, neuroexocytosis is impaired, a defect that can be rescued at a lower permissive temperature. We reveal that Munc18-1 undergoes K48-linked polyubiquitination, which is highly increased by the mutation, leading to proteasomal, but not lysosomal, degradation. Our data demonstrate that functional Munc18-1 levels are controlled through polyubiquitination and proteasomal degradation. The C180Y disease-causing mutation greatly potentiates this degradative pathway, rendering Munc18-1 unable to facilitate neuroexocytosis, a phenotype that is reversed at a permissive temperature.
[Show abstract][Hide abstract] ABSTRACT: Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.
Full-text · Article · Sep 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Protein dimerisation and oligomerisation is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to information transfer in signalling, transcriptional and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allows rapid mapping of homo and hetero oligomerisation of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerisation of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pairwise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerisation partners for each family member. We find that while nine SNX-BAR proteins are able to form homodimers, all members including the retromer-associated SNX1, SNX2 and SNX5 can form selective heterodimers, while SNX2, SNX4, SNX6 and SNX8 are promiscuous binders of other SNX-BAR proteins. Remarkably, we also observed that the BAR domain lacking SNX3 interacts with SNX8 indicating a different interaction mode. We conclude that the presented combination of methods enables rapid reconstitution and analysis of protein-protein interaction networks in vitro.
No preview · Article · May 2014 · Molecular & Cellular Proteomics
[Show abstract][Hide abstract] ABSTRACT: Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription
factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is
enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine
receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of
the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control
the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures
of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of
the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel
TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the
kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well
generalize to other class I cytokine receptors.
[Show abstract][Hide abstract] ABSTRACT: Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens
(ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated
with cadherin adhesion is not known. We now identify cortactin as a key scaffold for actin regulation at the ZA, which localizes
to the ZA through influences from both E-cadherin and N-WASP. Using cell-free protein expression and fluorescent single molecule
coincidence assays, we demonstrate that cortactin binds directly to the cadherin cytoplasmic tail. However, its concentration
with cadherin at the apical ZA also requires N-WASP. Cortactin is known to bind Arp2/3 directly (Weed, S. A., Karginov, A.
V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., and Parsons, J. T. (2000) J. Cell Biol. 151, 29–40). We further show that cortactin can directly bind WAVE2, as well as Arp2/3, and both these interactions are necessary
for actin assembly at the ZA. We propose that cortactin serves as a platform that integrates regulators of junctional actin
assembly at the ZA.
Full-text · Article · Jan 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: In this chapter, we describe the production and application of a eukaryotic cell-free expression system based on Leishmania tarentolae. This single-celled flagellate allows straightforward and inexpensive cultivation in flasks or bioreactors. Unlike many other Leishmania species, it is nonpathogenic to humans and does not require special laboratory precautions. An additional reason it is a convenient source organism for cell-free lysate production is that all endogenous protein expression can be suppressed by a single antisense oligonucleotide targeting splice leader sequence on the 5'-end of all protein coding RNAs. We describe simple procedures for cell disruption and lysate processing starting from bioreactor culture. We also describe introduction of genetic information via vectors containing species-independent translation initiation sites (SITS). We consider that such an inexpensive eukaryotic cell-free production system has many advantages when expressing multi-subunit proteins or difficult to express proteins.
No preview · Article · Jan 2014 · Methods in molecular biology (Clifton, N.J.)