The production of recombinant proteins containing unnatural amino acids, commonly known as genetic code expansion (GCE), represents a breakthrough in protein engineering that allows for the creation ...of proteins having novel designed properties. The naturally occurring orthogonal pyrrolysine tRNA/aminoacyl-tRNApyl synthetase pair (tRNApyl/PylRS) found in Methanosarcinaceae species has provided a rich platform for protein engineers to build a library of amino acid derivatives suitable for the introduction of novel chemical functionalities. While reports of the production of such recombinant proteins utilizing the tRNApyl/PylRS pair, or mutants thereof, is commonplace in Escherichia coli and mammalian cell expression systems, there has only been a single such report of GCE in the other stalwart of recombinant protein production, the baculovirus expression vector system (BEVS). However, that report formulates protein production within the designs of the MultiBac expression system 1. The current study frames protein production within the strategies of the more commonplace Bac-to-Bac system of recombinant baculovirus production, via the development of novel baculovirus transfer vectors that harbor the tRNApyl/PylRS pair. The production of recombinant proteins harboring an unnatural amino acid(s) was examined using both an in cis and an in trans arrangement of the tRNApyl/PylRS pair relative to the target protein ORF i.e. the latter resides, respectively, on either the same vector as the tRNApyl/PylRS pair, or on a separate vector and deployed in a viral co-infection experiment. Aspects of the transfer vector designs and the viral infection conditions were investigated.
•Bac-to-Bac compatible transfer vectors were designed for genetic code expansion in baculovirus-infected insect cells (BEVS).•Novel Bac-to-Bac compatible transfer vectors demonstrated successful incorporation of unnatural amino acids.•The production of proteins containing unnatural amino acids was optimized for insect cells using BEVS.•First report using the Bac-to-Bac system of recombinant baculovirus production for genetic code expansion.
The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) ...has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.
In the past 20 years, the use of ultrasound-based methods has become a standard approach to measure tendon mechanical properties in vivo. Yet the multitude of methodological approaches adopted by ...various research groups probably contribute to the large variability of reported values. The technique of obtaining and relating tendon deformation to tensile force in vivo has been applied differently, depending on practical constraints or scientific points of view. Divergence can be seen in 1) methodological considerations, such as the choice of anatomical features to scan and to track, force measurements, or signal synchronization; and 2) in physiological considerations related to the viscoelastic behavior or length measurements of tendons. Hence, the purpose of the present review is to assess and discuss the physiological and technical aspects connected to in vivo testing of tendon mechanical properties. In doing so, our aim is to provide the reader with a qualitative analysis of ultrasound-based techniques. Finally, a list of recommendations is proposed for a number of selected issues.
Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent ...and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis. We report here the crystal structures of apo-PTK6 and inhibitor-bound PTK6 complexes, providing the structural basis for small molecule interaction with PTK6. The kinase inhibitors moderately suppress tumor cell growth in 2D and 3D cell cultures. However, the tumor cell growth inhibition shows neither correlation with the PTK6 kinase activity inhibition, nor the total or activated PTK6 protein levels in tumor cells, suggesting that the tumor cell growth is independent of PTK6 kinase activity. Furthermore, in engineered breast tumor cells overexpressing PTK6, the inhibition of PTK6 kinase activity does not parallel the inhibition of tumor cell growth with a >500-fold shift in compound potencies (IC50 values). Overall, these findings suggest that the kinase activity of PTK6 does not play a significant role in tumorigenesis, thus providing important evidence against PTK6 kinase as a potential therapeutic target for breast cancer treatment.
Recombinant protein production in the baculovirus expression vector system (BEVS) has emerged as a system of choice for the production of recombinant human proteins for R&D purposes. Scale-up protein ...production in insect cells past the one or two liter volume generally utilizes disposable cellbag bioreactors that provide a means to scale to the 5-25L range in a single vessel. However, cellbags can be expensive and their use requires capital investment in dedicated rocker platforms and their associated air pumps and exhaust heaters. Additional equipment, such as tube welders and liquid pumps are often also deployed for the sterile transfer of media outside of a biosafety cabinet. Herein it is reported that Sf9, Sf21 and High Five insect cells demonstrate normal growth characteristics when cultured at the 2.5 L level in 3 L Erlenmeyer flasks, or at the 4.5 L level in 5 L Erlenmeyer flasks in standard laboratory shakers. In addition, a direct comparison of the expression levels of four separate proteins at the 4.5 L scale in 5 L flasks versus those at the 5 L scale in 10 L cellbags demonstrates that protein production is equal to, or slightly better, in the flasks versus the cellbags. The adoption of high-volume shake flasks for routine recombinant protein production in insect cells has a number of advantages over disposable bioreactors in terms of ease of use, and equipment and disposables costs.
•Sf9, Sf21 and High Five insect cells demonstrate normal growth characteristics at the 4.5 L level in 5 L Erlenmeyer flasks.•Recombinant protein expression in Sf21 insect cells at 4.5L in a 5L Erlemeyer flask is equivalent to using 5L in a 10L cellbag.•High-volume shake flask protein expression culture in insect cells has advantages over cellbag technology.
Recent studies suggest region‐specific metabolic activity in hamstring muscles during injury prevention exercises, but the neural representation of this phenomenon is unknown. The aim of this study ...was to examine whether regional differences are evident in the activity of biceps femoris long head (BFlh) and semitendinosus (ST) muscles during two common injury prevention exercises. Twelve male participants without a history of hamstring injury performed the Nordic hamstring exercise (NHE) and stiff‐leg deadlift (SDL) while BFlh and ST activities were recorded with high‐density electromyography (HD‐EMG). Normalized activity was calculated from the distal, middle, and proximal regions in the eccentric phase of each exercise. In NHE, ST overall activity was substantially higher than in BFlh (d = 1.06 ± 0.45), compared to trivial differences between muscles in SDL (d = 0.19 ± 0.34). Regional differences were found in NHE for both muscles, with different proximal‐distal patterns: The distal region showed the lowest activity level in ST (regional differences, d range = 0.55‐1.41) but the highest activity level in BFlh (regional differences, d range = 0.38‐1.25). In SDL, regional differences were smaller in both muscles (d range = 0.29‐0.67 and 0.16‐0.63 in ST and BFlh, respectively) than in NHE. The use of HD‐EMG in hamstrings revealed heterogeneous hamstrings activity during typical injury prevention exercises. High‐density EMG might be useful in future studies to provide a comprehensive overview of hamstring muscle activity in other exercises and high‐injury risk tasks.
Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen ...the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro. In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17–3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer–monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.
SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and ...is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.
The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer ...that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.
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