Dwight Nissley - Academia.edu (original) (raw)

Papers by Dwight Nissley

This article cites 38 articles, 23 of which can be accessed free

Cancer informatics, 2017

The 3 human RAS genes play pivotal roles regulating proliferation, differentiation, and survival ... more The 3 human RAS genes play pivotal roles regulating proliferation, differentiation, and survival in normal cells and become mutated in 15% to 20% of all human tumors and amplified in many others. In this report, we examined data from The Cancer Genome Atlas to investigate the relationship between RAS gene mutational status and messenger RNA expression. We show that all 3 RAS genes exhibit increased expression when they are mutated in a context-dependent manner. In the case of KRAS, this increase is manifested by a larger proportional increase in KRAS4A than KRAS4B, although both increase significantly. In addition, the mutational status of RAS genes can be associated with expression changes in other RAS genes, with most of these cases showing decreased expression. The mutational status associations with expression are recapitulated in cancer cell lines. Increases in expression are mediated by both copy number variation and contextual differences, including mutational status of epide...

Cancer Research, 2016

KRAS is the most frequently mutated member of RAS superfamily in all human cancers. Post-translat... more KRAS is the most frequently mutated member of RAS superfamily in all human cancers. Post-translational farnesylation and methylation of KRAS at its C-terminal end plays an important role in its trafficking to proper subcellular compartments for cell signaling events. PDEδ acts as a chaperone for farnesylated members of the RAS superfamily and plays a key role in their trafficking from the place of biosynthesis or post-translational modification to a transport vesicle or plasma membrane. We solved the structures of wild-type full-length KRAS4b (farnesylated and methylated) in complex with PDEδ in two different crystal forms at 2 Ang resolution. Our structure showed that only the last six amino acids of hypervariable region (and not the G-domain) of KRAS4b interact with PDEδ. Farnesyl and methyl groups present on Cys185 bind tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we could see all amino acids present in the hypervariable region of KRAS4b, thus pr...

Biomarkers in medicine, 2014

The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteom... more The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear c...

Biophysical Journal, 2019

Molecular diffusion plays a fundamental role in a vast array of biological processes. Brownian dy... more Molecular diffusion plays a fundamental role in a vast array of biological processes. Brownian dynamics is a computational simulation technique used to model the diffusional encounter of two molecules in solution. It can be used to estimate second-order rate constants, calculate reaction probabilities, and report reaction trajectories. Here, we applied such methods to explore two time-consuming diffusional processes: substrate binding to a protein and intermediate channeling among enzymes. The objective of the former study is to understand the details of molecular encounter that may play a role in the efficient operation of the 3 0-5 0-cyclic adenosine monophosphate (cAMP) signaling apparatus which is terminated by phosphodiesterases (PDEs). The latter study focuses on the mechanistic basis of oxaloacetate (OAA) channeling within possible malate dehydrogenase-citrate synthase (MDH-CS) metabolons that have different structural orientations in their complexes. We reported that the electrostatic potential of the active site from PDE and an electrostatic channel formed in the enzyme metabolons guide the oppositely-charged substrate molecules toward the target spots. This electrostatic steering promotes the capture of diffusing substrates and is of significance in sequestering intermediates released from a nearby binding site or in attracting more freely diffusing molecules to enhance the binding and transfer efficiency.

We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the... more We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae. The RT activity of these hybrid Ty1yHIV-1 (his3AIyAIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (2)-(S)-8-Chloro4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are ...

Heidmann et al., "Retransposition of a Mouse LAP Sequence Tagged With an Indicator Gene&quot... more Heidmann et al., "Retransposition of a Mouse LAP Sequence Tagged With an Indicator Gene" Cell 84: 159-170 (Jan. 1991). Garfinkel, D. et al., "Transposon Tagging Using Ty Elements in Yeast." Genetics 120: 95-108 (Sep. 1988). Boeke, J.D. et al., "A General Method for the Chromosomal Amplification of Genes in Yeast"Science 239:280-282 (Jan. 1988). Boeke, J.D. et al., “Ty Elements Transpose Through an RNA Intermediate" Cell 40:491-500 (Mar. 1985). III US005714313A

Tipifarnib, a drug that targets HRAS through inhibiting farnesyl transferase, is in phase II clin... more Tipifarnib, a drug that targets HRAS through inhibiting farnesyl transferase, is in phase II clinical trials and appears to show activity in tumors harboring oncogenic HRAS mutations (https://kuraoncology.com/pipeline/#tipifarnib). Unfortunately, these drugs are not expected to be effective on KRAS cancers because KRAS, unlike HRAS, can be prenylated by geranylgeranyl transferase following farnesyl transferase inhibition. To address this, we have developed compounds that prevent farnesylation of KRAS 4B by covalent reaction with C185, the site of prenylation. These compounds bind to a pocket in the G-domain that is formed by interaction with the hypervariable region, an interaction that does not seem to occur in other RAS proteins. This existence of this pocket has been demonstrated through biochemical and biophysical analysis, including NMR and small-angle X-ray scattering. The compounds we have developed are active in cells: they prevent proliferation of MEFs driven by oncogenic K...

Cell Reports, 2020

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogenactivated... more Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogenactivated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPaseactivating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105,

RAS is a signaling protein associated with the cell membrane that is mutated in 30% of human canc... more RAS is a signaling protein associated with the cell membrane that is mutated in 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influen...

ABSTRACTA vital first step of RAF activation involves binding to active RAS, resulting in the rec... more ABSTRACTA vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Journal of Biological Chemistry, 2020

The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutantactive R... more The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutantactive RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 Å resolutions, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.

Clinical Research (Excluding Clinical Trials), 2020

Traditional proteomic methods face an inherent challenge with the RAS family of GTPases (HRAS, NR... more Traditional proteomic methods face an inherent challenge with the RAS family of GTPases (HRAS, NRAS, KRAS4A, KRAS4B). Expressed at low endogenous abundance, these four isoforms possess considerable sequence similarity, rendering the confident proteomic linkage of a given isoform with a specific mutation or post-translational modification (PTM) a formidable proposition outside of isoform-specific model cell lines. Conversely, top-down proteomic analysis (TDP), which measures intact protein molecules, can precisely identify modified protein forms, or proteoforms, and confidently localize both mutations and PTMs on each RAS isoform present. Previously, a method combining immunoprecipitation and subsequent TDP (IP-TDP) led to the identification of eleven KRAS4B proteoforms, including four mutations and two novel PTMs, within colorectal cancer cell lines and tumor samples†. These initial results demonstrated the great potential for proteoform-resolved measurements to further illuminate the role of KRAS4B mutations and modifications in human cancer. More recently, the NCI RAS Initiative has developed an enhanced RAS proteoform assay incorporating IP-TDP analysis on an Orbitrap Fusion Lumos mass spectrometer. By employing alternate antibodies, recombinant protein standards, and well-characterized model or malignant cell lines, we have obtained substantial improvements in both RAS proteoform detection and characterization, thus facilitating the identification and localization of low-abundance PTMs. Moreover, we have applied our optimized IP-TDP method to a diverse panel of malignant and RAS mutant cell lines with the goal of expanding our knowledge of the RAS proteoform landscape. In doing so, we have observed a wealth of novel RAS proteoforms, of far greater number and complexity than previously anticipated. We have also observed isoform-specific proteoform variations, most notably between KRAS4A and KRAS4B, and initial indications of proteoform context dependence, namely within a given mutation or tissue background. Our results further underscore the power of proteoform-resolved measurements and indicate that RAS-dependent signaling pathways in normal and cancer contexts may be far more complex than previously thought. † I Ntai et. al. “Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification crosstalk.” Proc Natl Acad Sci U S A. 2018 Apr 17; 115(16):4140-4145. Citation Format: Caroline DeHart, Kanika Sharma, Dominic Esposito, Anna Maciag, Dwight Nissley, Frank McCormick. Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5133.

Proceedings of the International Conference for High Performance Computing, Networking, Storage and Analysis, Nov 17, 2019

are no longer affiliated with the respective institutions.

Biophysical Journal, 2020

Acta Crystallographica Section A Foundations and Advances, 2019