Dekker F.J.,Max Planck Institute of Molecular Physiology |
Dekker F.J.,University of Marburg |
Dekker F.J.,University of Groningen |
Rocks O.,Max Planck Institute of Molecular Physiology |
And 25 more authors.
Nature Chemical Biology | Year: 2010
Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localizationĝ€"and thereby unregulated signalingĝ€"caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.
Stalnecker C.A.,Baker Laboratory |
Erickson J.W.,Baker Laboratory |
Cerione R.A.,Baker Laboratory |
Cerione R.A.,Cornell University
Journal of Biological Chemistry | Year: 2017
The first step in glutamine catabolism is catalysis by the mitochondrial enzyme glutaminase, with a specific isoform, glutaminase C (GAC), being highly expressed in cancer cells. GAC activation requires the formation of homotetramers, promoted by anionic allosteric activators such as inorganic phosphate. This leads to the proper orientation of a flexible loop proximal to the dimer-dimer interface that is essential for catalysis (i.e. the "activation loop"). A major class of allosteric inhibitors of GAC, with the prototype being bis-2-(5-phenylacetamido-1,2,4-Thiadiazol-2-yl)ethyl sulfide (BPTES) and the related molecule CB-839, binds to the activation loop and induces the formation of an inactive tetramer (two inhibitors bound per active tetramer). Here we describe a direct readout for monitoring the dynamics of the activation loop of GAC in response to these allosteric inhibitors, as well as allosteric activators, through the substitution of phenylalanine at position 327 with tryptophan (F327W). The tryptophan fluorescence of the GAC(F327W) mutant undergoes a marked quenching upon the binding of BPTES or CB-839, yielding titration profiles that make it possible to measure the binding affinities of these inhibitors for the enzyme. Allosteric activators like phosphate induce the opposite effect (i.e. fluorescence enhancement). These results describe direct readouts for the binding of the BPTES class of allosteric inhibitors as well as for inorganic phosphate and related activators of GAC, which should facilitate screening for additional modulators of this important metabolic enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Finkelstein D.A.,Baker Laboratory |
Kirtland J.D.,Cornell University |
Mota N.D.,Baker Laboratory |
Stroock A.D.,Cornell University |
Abruna H.D.,Baker Laboratory
Journal of Physical Chemistry C | Year: 2011
Oxygen (O2) reduction has long been the factor limiting the power density of most fuel cells. Membraneless, microfluidic fuel cells are a promising new fuel cell technology, yet they are affected even more strongly by O2, as they usually require a dissolved oxidant, and O2 has minimal solubility in most solvents. Here we offer a detailed, analytical comparison of the performance of previously employed alternative oxidants, H2O2, MnO4 ?, VO2 +, and ClO?, at Pt, Au, and glassy carbon (GC) rotating disk electrodes (RDEs). We also investigated cerium ammonium nitrate (CAN), which has an exceptionally high potential for reduction. Of the oxidants studied, CAN offers the best immediate advantage, but MnO4 ?, though requiring development efforts, shows the most long-term promise for high-power fuel cells. © 2011 American Chemical Society.
Booth J.G.,Cornell University |
Eilertson K.E.,Cornell University |
Olinares P.D.B.,Baker Laboratory |
Olinares P.D.B.,Cornell University |
Yu H.,Cornell University
Molecular and Cellular Proteomics | Year: 2011
Recent developments in mass-spectrometry-based shotgun proteomics, especially methods using spectral counting, have enabled large-scale identification and differential profiling of complex proteomes. Most such proteomic studies are interested in identifying proteins, the abundance of which is different under various conditions. Several quantitative methods have recently been proposed and implemented for this purpose. Building on some techniques that are now widely accepted in the microarray literature, we developed and implemented a new method using a Bayesian model to calculate posterior probabilities of differential abundance for thousands of proteins in a given experiment simultaneously. Our Bayesian model is shown to deliver uniformly superior performance when compared with several existing methods. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.