Biology Faculty: Dan Fabris

Dan Fabris

Professor of Chemistry and Biological Sciences
Ph.D., University of Padua

Office LS1109
Telephone (518) 437-4464
Fax (518) 442-3462
Email fabris@albany.edu

Areas of Interest

  • Using Mass spectroscopy to investigate macromolecular complexes
  • Protein-nucleic acid interactions in viruses
  • High-resolution mass spectrometry
  • RNA-based drug therapeutics




Research

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Our laboratory is dedicated to the investigation of the structure-function relationships of protein-nucleic acid complexes involved in the lifecycle of viruses responsible for infectious diseases, using mass spectrometry (MS) as the main instrumental platform. The greatest discoveries in biology and medicine have always been the fruit of significant technological advances and, vice versa, the greatest technological breakthroughs have always been prompted by the need to raise to the challenge of the biomedical problems of our times. The two go hand in hand in an indissoluble bond. It is this belief that permeates our research.

An example is provided by our investigation of human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS. In this retrovirus, the 5’-untranslated region (5’-UTR) governs key replication steps in which viral RNA acts either as genome intended for packaging, or as mRNA meant for protein expression. The functions associated with these distinct spheres of activities are controlled by signals included in discrete elements of secondary structure, which define the overall 3D structure by establishing mutual tertiary interactions. In vitro experiments have provided evidence that 5’-UTR may assume different folds characterized by significant rearrangement of secondary structure, tertiary interactions, and overall architecture. Transitions between conformers are facilitated in vitro by the chaperone activity of the nucleocapsid (NC) domain of the Gag polyprotein, which is known for promoting remodeling of nucleic acid structure. These observations have prompted the hypothesis that NC/Gag may represent the actuator of a riboswitch mechanism that coordinates the multifaceted activities of 5'-UTR by exposing different signals at different points of the virus lifecycle. This hypothesis presupposes a process of specific recognition between unique structures present in the alternative conformers and cognate viral/host factors involved in the various activities, but the determinants of such interactions are still not understood. A variety of approaches including phylogenetic analysis, site-directed mutagenesis, knockdown experiments, and other biological assays have demonstrated different levels of functional correlation between 5'-UTR signals and cognate proteins. Explaining these functional relationships, however, has been hampered by the absence of actual structural data for pertinent protein-RNA assemblies implicated in such functions, which is largely due to the challenges of investigating these substrates directly in their natural environment. Despite extensive efforts, the dearth of in vivo structural information has limited our understanding of the determinants driving the recognition of specific viral/host factors, thus hampering their utilization as possible targets for rational drug design.

We propose that the missing information could be obtained by combining chemical probing approaches with mass spectrometry detection (MS). Treating target substrates with these types of reagents resembles exposing photographic film to light. The process leaves a permanent “impression” of the substrate structure in the form of a modification pattern specific to the 3D fold, which can be subsequently “developed” by any suitable means that do not necessarily have to preserve the original fold. Taking advantage of this favorable characteristic, we are exploring the application of structural probing techniques to viral particles and infected cells. Once virions/cells have been exposed to the probe, they can be lysed to initiate the "development" process through experimental procedures that may employ denaturing conditions. For example, the target ribonucleoprotein can be extracted by affinity capture using beads that are coated with suitable antisense oligonucleotides. The captured RNA can undergo cleavage by specific deoxyribozymes to isolate the region of interest. The probed products can be finally characterized by mass mapping and sequencing technologies. The MS platform is directly applicable to virtually all biomolecules and their mixtures, affording numerous advantages over techniques that require chromophores or probe-specific cleavage. The fact that the elemental composition of intact molecular ions and corresponding gas-phase fragments are very characteristic for each class of biopolymers and modifiers, alike, translates into the ability of achieving their full and unambiguous characterization from their unique mass signatures. We have been exploring MS approaches to facilitate the analysis of RNA-RNA and protein-RNA conjugates produced by bifunctional crosslinkers, which offer information about the spatial relationships between bound components. We are devising approaches capable of providing a direct view of binding interfaces, which are based on gas-phase processes rather than on probe modifications. We are also engaged in the development of software tools that support the interpretation of these types of data and enable the effective utilization of the corresponding spatial constraints in molecular modeling operations. Recent advances by computational techniques in structural biology have helped close the resolution gap by providing atomic-level details that are typically beyond the reach of chemical probes, thus dramatically increasing the value and reach of this type of approach for structural determination. We are taking advantage of these advances to pursue the elucidation of 5'-UTR and its functional ribonucleoproteins directly in their natural environments, which could provide the keys to understanding the mechanism by which this system performs its critical biological activities.

At the same time, we have been exploring critical components of HIV and analogous retroviral systems as possible targets for the development of small ligand inhibitors. The possibility of detecting intact non-covalent interactions by electrospray ionization (ESI) mass spectrometry has presented us with the opportunity to investigate the binding properties of chemicals that lack the chromophores necessary for their spectroscopic detection. Taking advantage of this favorable feature, we have developed technologies for determining the stoichiometry and binding affinity of ligands toward RNA substrates. We found new ways for determining the concentration of ligand that induces 50% inhibition of a target protein-RNA complex (i.e., IC50). The high resolution afforded by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has allowed us to investigate multiple ligands simultaneously in competitive binding experiments that can immediately identify the tightest binder in the pool and rank the relative binding affinities of the remaining ones. In proof-of-principle experiments, we created a random combinatorial library containing ~45,000 hepta-peptides, which was fractionated in three pools based on solubility in water, methanol, and 1:1 water:methanol. Each pool was then screened against a mixture of four selected structures of HIV-1 5'-UTR, which revealed the presence of ~50 species with superior binding affinity toward the different targets. These operations required ~30 min and resulted in reducing the number of potential drug candidates to the point where expensive in vivo screening becomes economically viable. We are now pursuing the implementation of front-end robotics and microfluidics systems to enable unattended high-throughput operations, which will allow us to explore a wider swath of chemical space for promising compounds. We believe that only by developing this and other novel technologies will it be possible to accelerate the pace of drug discovery to keep up with emergence of new infectious diseases and the selection of drug-resistant strains.

Publications

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  • Fabris D, Limbach PA. (2010) Review of the 22nd Sanibel Conference on Mass Spectrometry: From Structural Biology to Drug Discovery: New Roles for Mass Spectrometry of Nucleic Acids. J Am Soc Mass Spectrom. Mar 6. [Epub ahead of print]
  • Fabris D. (2010) A role for the MS analysis of nucleic acids in the post-genomics age. J Am Soc Mass Spectrom. 21(1):1-13.
  • Turner KB, Yi-Brunozzi HY, Brinson RG, Marino JP, Fabris D, Le Grice SF. (2009) SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids. RNA 15(8):1605-13.
  • Turner KB, Yi-Brunozzi HY, Le Grice SF, Fabris D, Marino JP. (2009) Probing anomalous structural features in polypurine tract-containing RNA-DNA hybrids with neomycin B. Brinson RG, Biochemistry. 48(29):6988-97.
  • Ramos I, Fabris D, Qi W, Fernandez EJ, Good TA. (2009) Kinetic study of beta-amyloid residue accessibility using reductive alkylation and mass spectrometry. Biotechnol Bioeng. 104(1):181-92.
  • Gapeev A, Berton A, Fabris D. (2009) Current-controlled nanospray ionization mass spectrometry. J Am Soc Mass Spectrom. 20(7):1334-41.
  • Fabris D. (2009) Special Series: advances in the investigation of the structure-function relationship in nucleic acids and their assemblies. Biopolymers. 91(6):v.
  • Fabris D. (2009) Special Series: Advances in the investigation of the structure-function relationship in nucleic acids and their assemblies. Biopolymers. 91(4):iii-iv.
  • Turner KB, Kohlway AS, Hagan NA, Fabris D. (2009) Noncovalent probes for the investigation of structure and dynamics of protein-nucleic acid assemblies: the case of NC-mediated dimerization of genomic RNA in HIV-1. Biopolymers 91(4):283-96.
  • Yu ET, Hawkins A, Kuntz ID, Rahn LA, Rothfuss A, Sale K, Young MM, Yang CL, Pancerella CM, Fabris D. (2008) The collaboratory for MS3D: a new cyberinfrastructure for the structural elucidation of biological macromolecules and their assemblies using mass spectrometry-based approaches. J Proteome Res., 7(11):4848-57.
  • Legiewicz M, Badorrek CS, Turner KB, Fabris D, Hamm TE, Rekosh D, Hammarskjöld ML, Le Grice SF. (2008) Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element. Proc Natl Acad Sci U S A., 105(38):14365-70.
  • Turner, K. B.; Brinson, R. G.; Yi-Brunozzi, H. Y.; Rausch, J. W.; Miller, J. T. Le Grice, S. F. J.; Marino, J. P.; Fabris, D. (2008) “Recognition of HIV-1 Polypurine Tract RNA:DNA Hybrid by Classic Nucleic Acid Ligands” Nucleic Acids Res. 36, 2799-2810.
  • Zhang, Q.; Crosland, E.; Fabris, D. (2008) “Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies” Anal Chim Acta. 627(1):117-28.
  • Yu ET, Hawkins A, Eaton J, Fabris D. (2008) MS3D structural elucidation of the HIV-1 packaging signal. Proc Natl Acad Sci USA. 105(34):12248-53.
  • Holland R, Hawkins AE, Eggler AL, Mesecar AD, Fabris D, Fishbein JC.(2008) Prospective type 1 and type 2 disulfides of Keap1 protein. Chem Res Toxicol. 21(10):2051-60.
  • Hagan, N. A. and Fabris, D. (2007) “Dissecting the protein-RNA and RNA-RNA interactions in the nucleocapsid-mediated dimerization and isomerization of HIV-1 stemloop 1” J. Mol. Biol. 365, 396-410 .
  • Fabris, D.; Chaudhari, P.; Hagan, N. A.; Turner, K. B. (2007) “Functional Investigations of Retroviral Protein-RNA Complexes by Nanospray-FTICR Mass Spectrometry” Eur. J. Mass Spectrom. 13, 29-33 .
  • Turner, K. B.; Hagan, N. A.; Fabris, D. (2007) “Understanding the isomerization of the HIV-1 dimerization initiation domain by the nucleocapsid protein” J. Mol. Biol. 369, 812-828 .
  • Stephen, A. G.; Datta, S. A. K.; Worthy, K. M.; Bindu, L.; Fivash, M.J.; Turner, K. B.; Fabris, D.; Rein, A.; Fisher, R.J. (2007) “Measuring the binding stoichiometry of HIV-1 Gag to very-low-density oligonucleotide surfaces using plasmon resonance spectroscopy” J. Biomol. Tech. 18, 259-266 .
  • Turner, K. B.; Hagan, N. A..; Fabris, D. (2006) “Inhibitory effects of archetypical nucleic acid ligands on the interactions of HIV-1 nucleocapsid protein with elements of Y-RNA”, Nucleic Acids Res. 34, 1305-1316 .
  • Fisher, R.J.; Fivash, M.J.; Stephen, A.G.; Hagan, N.A.; Shenoy, S.R.; Medaglia, M.V.; Smith, L.R.; Worthy, K.M.; Simpson, J.T.; Shoemaker, R.; McNitt, K.L.; Johnson, D.J.; Hixson, C.V.; Gorelick, R.J.; Fabris, D.; Henderson, L.E.; Rein, A. (2006) “Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides”, Nucleic Acids Res. 34, 472-484 .
  • Rait, V. K.; Zhang, Q.; Fabris, D.; Mason, J. T.; O’Leary, T. J. (2006) “Conversions of formaldehyde-modified 2´-deoxyadenosine 5´-monophosphate in conditions modeling formalin-fixed tissue dehydration” J. Histochem. Cytochem. 54, 301-310 .
  • Turner, K. B.; Hagan, N. A.; Kohlway, A.; Fabris, D. (2006) “Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry” J. Am. Soc. Mass Spectrom. 17, 1401-1411 , journal cover.
  • Zhang, Q.; Yu, E. T.; Kellersberger, K. A.; Crosland, E.; Fabris, D. (2006) “Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures” J. Am. Soc. Mass Spectrom. 17, 1570-1581 .

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