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Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

Study Design Retrospective Cohort Objective Antiresorptive drugs are often given to minimize fracture risk for bone metastases, but data regarding optimal time or ability to reduce stereotactic body radiotherapy (SBRT)-induced fracture risk is limited. This study examines the association between antiresorptive use surrounding spinal SBRT and vertebral compression fracture (VCF) incidence to provide information regarding effectiveness and optimal timing of use. Methods Patients treated with SBRT for spinal metastases at a single institution between 2009-2020 were included. Kaplan-Meier analysis was used to compare cumulative incidence of VCF for those taking antiresorptive drugs pre-SBRT, post-SBRT only, and none at all. Cox proportional hazards and Fine-Gray competing risk models were used to identify additional factors associated with VCF. Results Of the 234 patients (410 vertebrae) analyzed, 49 (20.9%) were taking bisphosphonates alone, 42 (17.9%) were taking denosumab alone, and 25 (10.7%) were taking both. Kaplan-Meier analysis revealed a statistically significant lower VCF incidence for patients initiating antiresorptive drugs before SBRT compared to those taking none at all (4% vs 12% at 1 year post-SBRT, P = .045; and 4% vs 23% at 2 years, P = .008). On multivariate analysis, denosumab duration (HR: .87, P = .378) or dose (HR: 1.00, P = .644) as well as bisphosphonate duration (HR: .98, P= .739) or dose (HR: .99, P= .741) did not have statistical significance on VCF incidence. Conclusion Initiating antiresorptive agents before SBRT may reduce the risk of treatment-induced VCF. Antiresorptive drugs are underutilized in patients with spine metastases and may represent a useful intervention to minimize toxicity and improve long-term outcomes.

Millions of people a year receive magnetic resonance imaging (MRI) contrast agents for the diagnosis of conditions as diverse as fatty liver disease and cancer. Gadolinium chelates, which provide preferred T

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein that is required for successful long-term memory formation. Ca2+/CaM activates CaMKII by binding to its regulatory segment, thereby making the substrate binding pocket available. One exceptional feature of this kinase is that two binding partners have been shown persistently activate CaMKII after the Ca2+ stimulus dissipates. The molecular details of this phenomenon are unclear. Despite having a large variety of interaction partners, the specificity of CaMKII has not been structurally well-characterized. To this end, we solved X-ray crystal structures of the CaMKII kinase domain bound to four different binding partners: a peptide substrate, a substrate/activator, a substrate/binding partner, and an inhibitor (AMPA-type glutamate receptor, NMDA-type glutamate receptor, Tiam1, and Densin-180). We show that all four binding partners use similar interactions to bind across the substrate binding pocket of the CaMKII active site. We generated a sequence alignment based on our structural observations, which revealed conserved interactions across these binding partners. The structures presented here shed much-needed light on the interaction between CaMKII and its binding partners. These observations will be crucial in guiding further biological experiments.

Millions of people a year receive magnetic resonance imaging (MRI) contrast agents for the diagnosis of conditions as diverse as fatty liver disease and cancer. Gadolinium chelates, which provide preferred T1 contrast, are the current standard but face an uncertain future due to increasing concerns about their nephrogenic toxicity as well as poor performance in high-field MRI scanners. Gadolinium-containing nanocrystals are interesting alternatives as they bypass the kidneys and can offer the possibility of both intracellular accumulation and active targeting. Nanocrystal contrast performance is notably limited, however, as their organic coatings block water from close interactions with surface Gadoliniums. Here, these steric barriers to water exchange are minimized through shape engineering of plate-like nanocrystals that possess accessible Gadoliniums at their edges. Sulfonated surface polymers promote second-sphere relaxation processes that contribute remarkable contrast even at the highest fields (r1 = 32.6 × 10-3 m Gd-1 s-1 at 9.4 T). These noncytotoxic materials release no detectable free Gadolinium even under mild acidic conditions. They preferentially accumulate in the liver of mice with a circulation half-life 50% longer than commercial agents. These features allow these T1 MRI contrast agents to be applied for the first time to the ex vivo detection of nonalcoholic fatty liver disease in mice.

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens., The present study characterizes integrin and DDR2 signalling networks activated by collagen. Using clustering approaches, DDR2-specific signalling components such as SHP-2 were identified. We further demonstrate that SHP-2 is phosphorylated by a subset of DDR2 lung cancer mutants.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Receptor tyrosine kinases have become important therapeutic targets because of their involvement in diseases, including cancer. Kinase domains, which are soluble and easily purified, have found widespread use in enzyme inhibitor assays, but these domains do not exhibit full function because they are isolated from the membrane. To address this shortcoming, the authors developed a simple method to restore biologically relevant function by assembling kinase domains on a nanometer-scale template, which imitates the membrane surface. Autophosphorylation of template-assembled tyrosine kinase domains from the insulin, EphB2, and Tie2 receptors led to substantially larger phosphorylation levels compared with domains assayed under conventional conditions. Template-directed assembly increased the total substrate phosphorylation of the insulin and EphB2 receptor kinase domains as much as 60-fold and 15-fold, respectively. In contrast, substrate phosphorylation by template-assembled Tie2 was much lower than conventional conditions. The lower activity observed with the template is more biologically relevant because autophosphorylation of Tie2 is self-inhibitory. These results, as well as the underlying similarity between the organization of template-assembled and natural membrane signaling environments, suggest that template-directed assembly of signaling proteins will provide widespread benefit to basic and applied signal transduction research, especially drug discovery. (Journal of Biomolecular Screening 2008:810-816)

A multitude of proteins reside at or near the cell membrane, which provides a unique environment for organizing and promoting assemblies of proteins that are involved in a variety of cellular signaling functions. Many of these proteins and pathways are implicated in disease. For example, strong links have been established between receptor tyrosine kinases and disease, most notably, cancer. However, a significant impediment to researchers remains: membrane-associated proteins are difficult to reconstitute and study. Template-directed assembly represents a powerful new technology that enables the assembly of membrane-associated proteins. We show that template-directed assembly restores tyrosine kinase activity and regulation, and provides a way for researchers to build multicomponent assemblies. As an example of better enzyme regulation, the Tie2 tyrosine kinase domain exhibits (biologically relevant) autoinhibitory behavior when template assembled. Also, template-assembled insulin receptor tyrosine kinase domains exhibit significant autophosphorylation (none detected without template-directed assembly) and an eightfold increase in substrate phosphorylation (compared to best solution conditions). Thus, template-directed assembly has a demonstrated ability to effectively produce more biologically relevant results using these commercial reagents. Template-directed assembly promises to be generally applicable to the signaling networks important for human health, because these pathways frequently contain membrane-associated proteins that require the organizing influence of a membrane surface.

RNA polymerases bind to specific sequences in DNA, melt open duplex DNA around the start site, and start transcription within the initially melted bubble. The initially transcribing complex is relatively unstable, releasing short abortive products. After synthesis of a minimal length of RNA (approximately 10-12 bases in the T7 system), RNA polymerases complete the transition to a processive (highly stable) elongation phase and lose the initial promoter contacts. The current study strongly supports a model for T7 RNA polymerase in which initial bubble collapse from position -4 to position +3 is responsible for initiating RNA displacement in the transition process. More specifically, collapse of the bubble from position -4 to position -1 indirectly and energetically facilitates the direct strand invasion offered by collapse at positions +1 to +3. Parallel work shows that promoter release, another key event occurring during this stage of transcription, begins after translocation to position +8 and is largely complete upon translocation to about position +12. The timing of promoter release agrees with the timing of initial bubble collapse determined by our previous fluorescence studies, suggesting that these two events are closely related.

Efficient recognition of DNA is a prerequisite for the development of biological effectors, including transcription and translation regulators, transfection vectors, and DNA sensors. To provide an effective scaffold for multivalent interactions with DNA, we have fabricated mixed monolayer protected gold clusters (MMPCs) functionalized with tetraalkylammonium ligands that can interact with the DNA backbone via charge complementarity. Binding studies indicate that the MMPCs and DNA form a charge-neutralized, nonaggregated assembly. The interactions controlling these assemblies are highly efficient, completely inhibiting transcription by T7 RNA polymerase in vitro.

In addition to its role as a barrier between the cytoplasm and the extracellular milieu, the cell membrane is a scaffold for a diverse collection of receptors and enzymes. The organization afforded by this scaffold serves to ensure an efficient interaction between the components of the membrane. The desire to maintain this organization in solution is a challenge for the appropriate interrogation of these biochemical components. This chapter will discuss strategies that allow biochemical analysis of membrane-associated enzymes within standard biochemical reactions. The advantages of these screening strategies in identifying valuable compounds from compound libraries and in understanding the intricacies of complex multiprotein complexes (i.e., chemotaxis) will be discussed.

Receptor tyrosine kinases have become important therapeutic targets because of their involvement in diseases, including cancer. Kinase domains, which are soluble and easily purified, have found widespread use in enzyme inhibitor assays, but these domains do not exhibit full function because they are isolated from the membrane. To address this shortcoming, the authors developed a simple method to restore biologically relevant function by assembling kinase domains on a nanometer-scale template, which imitates the membrane surface. Autophosphorylation of template-assembled tyrosine kinase domains from the insulin, EphB2, and Tie2 receptors led to substantially larger phosphorylation levels compared with domains assayed under conventional conditions. Template-directed assembly increased the total substrate phosphorylation of the insulin and EphB2 receptor kinase domains as much as 60-fold and 15-fold, respectively. In contrast, substrate phosphorylation by template-assembled Tie2 was much lower than conventional conditions. The lower activity observed with the template is more biologically relevant because autophosphorylation of Tie2 is self-inhibitory. These results, as well as the underlying similarity between the organization of template-assembled and natural membrane signaling environments, suggest that template-directed assembly of signaling proteins will provide widespread benefit to basic and applied signal transduction research, especially drug discovery. (Journal of Biomolecular Screening 2008:810-816)

Publisher Summary T7 RNA polymerase is an ideal model system in which to study fundamental characteristics of the complex machine that is RNA polymerase. Although structurally unrelated to the more complex multi‐subunit RNA polymerases, it appears that the common functional requirements imposed on all DNA dependent RNA polymerases is likely to be reflected in many common mechanistic features. Both the small size of the protein and the small size of the promoter DNA allow studies in the single subunit enzymes which are difficult or impossible in the much larger multi‐subunit enzymes. Similarly, the ability to prepare large amounts (and concentrations) of highly active T7 RNA polymerase opens doors that remain largely shut to the multi‐subunit systems. A self‐consistent picture of the critical transition from initiation to elongation is beginning to emerge. The resulting model presented here generates new testable hypotheses that will allow us to further refine our understanding of this complex molecular machine.

T7 RNA polymerase recognizes a small promoter, binds DNA, and begins the process of transcription by synthesizing short RNA products without releasing promoter contacts. To determine whether the promoter contact must be released to make longer RNA products and at what position the promoter must be released, a mutant RNA polymerase was designed that allows cross-linking to a modified promoter via a covalent disulfide bond. The modifications individually have no measurable effect on transcription. Under oxidizing conditions that produce the protein-DNA cross-link, the complex is able to synthesize short RNA products, strongly supporting a model in which promoter contacts are not lost on translocation through at least position +6. However, cross-linked complexes are impaired in promoter escape in that only about one in four can escape to make full-length RNA. The remainder release 12- and 13-mer RNA transcripts, suggesting an increased energetic barrier in the transition from an initial transcribing complex to a fully competent elongation complex. The results are discussed in the context of a model in which promoter release helps drive initial collapse of the upstream edge of the bubble, which, in turn, drives initial displacement of the 5'-end of the RNA.

Vascular endothelial growth factor (VEGF) is potent mitogen for endothelial cells that signals through VEGF Receptor-type2 (VEGFR-2)/Flk-1/KDR. Binding of VEGF to VEGFR-2, a membrane bound receptor tyrosine kinase of the VEGF-receptor family, leads to receptor dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling pathways stimulating angiogenesis. In adults, angiogenesis normally occurs during wound healing to restore blood flow post injury and during menstruation and pregnancy. Angiogenesis is also involved in several pathological disease states such as rheumatoid arthritis, psoriasis, diabetic retinopathy and cancer. While physiological angiogenesis involves interplay between pro- and anti-angiogenic factors, the ability to inhibit pathological angiogenesis has therapeutic potential. Inhibitors of VEGFR-2 provide targeted treatments for tumor growth as well as abnormal neovascularization related diseases. The caveat of traditional biochemical VEGFR-2 assays is that only the catalytic domain of the receptor is used to identify hits. One strategy to identify true novel inhibitors against membrane associated proteins is to screen these targets in a more physiological environment, where lipid-protein and protein-protein interactions are included in the assay. We achieved this goal by docking the entire cytoplasmic domain of the receptor on a liposome. Template Directed Assembly (TDA) is a screening technology designed to accomplish this objective. TDA technology utilizes recombinant VEGFR-2 cytoplasmic domain tagged with polyhistidine at the juxtamembrane position and docked to the liposome via the nickel residues. The lipids in TDA are able to diffuse within the surface of the liposome and, therefore, proteins can diffuse to form oligomeric complexes such as homo- or hetero-dimers formation. To compliment the TDA biochemical screen, we are implementing an image based high content screen (HCS) utilizing human umbilical vein endothelial cells (HUVECs) that express native VEGFR-2. The HCS screen is designed to identify potent and specific inhibitors of VEGFR-2 as assessed by a reduction in the proliferation marker Ki67. We are currently (1) validating the TDA assay with known VEGFR-2 inhibitors (2) screening a focus small molecule library to identify novel hits (3) testing the primary hits against a kinase selectivity panel and (4) designing and developing a VEGFR2 cell-based HCS assay to confirm the primary hits. The hit to lead program will be guided by using both physiologically relevant biochemical-based and cell-based assays. The combination of our novel TDA screening platform and the HCS assay technology permits us to identify unique VEGFR-2 inhibitors. Citation Format: Kelvin Lam, Edward Esposito, Deanna Navaroli, Ryan Clinton, Kerrin Beovich, Norman Garceau. Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2013-4544

Tropomyosin-related kinases (Trks A, B, and C) are a family of membrane-associated receptor tyrosine kinases (RTKs) activated by neurotrophins. RTKs are shown to be implicated in Alzheimer's disease, depression, pain sensation and various cancers including tumor cell growth and survival signaling. Inhibitors of Trk receptor kinases might provide targeted treatments for neurodegenerative diseases, pain and cancer. One strategy to indentify Trk isoform-specific inhibitors is to develop an assay where compound libraries can be screened in a more-native membrane associated lipid environment. Template-Directed Self Assembly (TDA) is a biomimetic technology designed to restore membrane-associated proteins to more relevant physiological conditions. This technology utilizes recombinant histidine-tagged proteins bound to the surface of nickel-chelated liposomes. Recombinant HIS-tagged proteins are engineered on the correct terminus of the protein to reflect the polarity of the enzyme with respect to the membrane. This allows the HIS-tagged proteins to bind to the liposome creating an environment much like a cellular membrane. The lipids in TDA are fully fluid within the surface of the liposome and associated proteins can rotate freely. Furthermore it promotes the formation of high-order structures such as homo- or hetro-dimers and shown to recruit accessory factors. Coupling of membrane associsted kinases to liposomes reconstitutes an environment that confers proper polarity, stereochemistry, topology and other critical properties, thereby creating a more relevant biochemical assay. To date, we have shown that several RTKs have enhanced biochemical specific activity and substrate specificity that resembles physiological conditions when coupled to TDA. The TDA approach restores TrkA activity to a more-native environment; therefore, we hypothesize that we will be able to identify compounds that are more specific and selective. To address the need of an enzyme assay near a lipid membrane environment, we successfully developed a robust TrkA assay shown to exhibit up to 20-fold increase in specific activity and a reduction in Km for ATP relative to TrkA in a conventional assay. Furthermore, TDA-associated TrkA exhibited a rapid and robust trans-activation, obviating the need for a pre-activation step. Thus, assays of TrkA coupled to TDA have the potential to identify more chemically diverse and specific inhibitors. We are currently (1) validating the TDA assay with known inhibitors (2) screening a focus library to identify novel hits (3) testing the primary hits against a small kinase selectivity panel and (4) designing and developing a TrkA specific reporter cell line to confirm the primary hits. The advantages of the TDA technology will be also discussed against other therapeutically relevant RTKs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2842. doi:1538-7445.AM2012-2842