Molecular basis of gene silencing

How do cells decide which genes to turn off and which genes to activate? The answer to this question partially lies in the spatial organization of the nucleus where cells sequester genes that need to be turned off in silencing compartments called heterochromatin, while active genes are organized in nuclear neighborhoods called euchromatin. We are interested in understanding how molecular decisions influence this global nuclear organization, and in turn, how nuclear compartmentalization influences gene regulation decisions on the molecular level. In particular, we focus on a family of proteins called heterochromatin proteins 1 (HP1). These proteins are key ingredients of silencing compartments and have interesting biophysical properties that allow them to play important roles in nuclear organization. To understand how these proteins work, we use a comprehensive toolbox of biophysical, chemical and cellular approaches including nuclear magnetic resonance (NMR), fluorescence imaging, in vitro biochemistry, and genetics. Understanding the functions and mechanisms of HP1 proteins has important implications for health and disease, in particular cancer, development disorders, and neurodegenerative disease.

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Molecular mechanisms of aging-related diseases

Debilitating aging-related diseases such as Alzheimer's involve the formation of pathological aggregates of proteins such as tau and Abeta. How these aggregates form in the complex cellular environment and how cells work to prevent aberrant aggregation is still not clear. We use nuclear magnetic resonance (NMR) spectroscopy, chemical biology, fluorescence imaging, biochemical and cellular approaches to follow the aggregation processes both in vitro and in cells. We are also interested in how cellular chaperones such as the small heat shock proteins (sHSPs) work to prevent or slow down aggregation, and how proteins such as tau may play a role in heterochromatin organization and gene regulation. In a recently funded international project, we also study the role of calcification in Alzheimer's disease and age-related macular degeneration (AMD), exploring the connection between the brain and the eye and the unexpected roles proteins such as tau and Abeta may play in the calcification process.

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Our methodologies

Nuclear magnetic resonance

Since most of the proteins we study have rigid and disordered parts and often adopt multiple dynamic states, we use a suite of NMR experiments to characterize their behavior, structure, dynamics, and interactions in vitro. This includes standard biomolecular solution NMR experiments, which allow us to characterize the smaller dynamic components of our biological systems, and a technique called solid-state magic angle spinning (MAS NMR) spectroscopy, which enables the characterization of large and more rigid systems including protein aggregates, chromatin polymers, chaperone-protein complexes, and protein-mineral deposits. We take advantage of our state-of-the-art NMR facility, which houses 600 MHz and 800 MHz NMR spectrometers for biomolecular solution NMR, as well as 750 MHz and 900 MHz NMR spectrometers for solid-state MAS NMR spectroscopy. The facility also houses a 600 MHz DNP NMR spectrometer, which allows unprecedented increase in sensitivity and the acquisition of NMR data from just a few micrograms of protein. This instrument has allowed us to develop a technique called SENiC (Sensitivity-Enhanced NMR in Cells), which enables the structural investigation of proteins in the cellular environment.

Chemical Biology

We take advantage of our chemical biology expertise to manipulate protein behavior both in vitro and in cells, in an effort to understand protein function and protein-protein interactions. For example, we use chemical approaches to install various post-translational modifications on our proteins of interest, as well as genetic encoding of non-canonical amino acids to target and manipulate proteins in cells. We also use inteins, proteins that are capable of splicing reactions, to prepare segmentally labeled proteins for NMR spectroscopy. Our SENiC approach relies heavily on the design and synthesis of DNP polarization agents, which we can target to specific proteins in cells, enhancing their NMR signals by several orders of magnitude and enabling structural biology in cells.

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800 MHz NMR spectrometer

600 MHz DNP NMR spectrometer

Galia Debelouchina - University of California, San Diego - Department of Chemistry and Biochemistry - © Debelouchina Lab 2024