Biochemical Characterization of Heterogeneous Nuclear Ribonucleoprotein A2 Inclusion Body Structure and Stability

Abstract

Inclusion bodies (IBs) are heterogeneous protein aggregates formed during recombinant protein expression in bacteria. IBs are advantageous for biotechnical applications as well as for research targeting the underlying mechanisms of protein aggregation in cells. Aberrant protein aggregation is central to various diseases, but the etiology and mechanisms remain poorly resolved in general. It is of great interest then to biochemically characterize disease-related aggregates in a tractable and controllable system. IBs are therefore an attractive model for examining how the cellular environment shapes aggregation, including the determinants relevant to pathological aggregation. The heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is found in various disease states, having aggregates discovered in postmortem patient tissue samples. hnRNPA2 is a ubiquitously expressed nucleic acid housekeeping protein in human cells, containing both structured domains and a prominent disordered prion-like domain. In particular, the intrinsically disordered low complexity domain (also referred to as the prion-like domain) of hnRNPA2 is the site of several point mutations that are associated with disease. For example, the D290V mutation is thought to aberrantly drive the naturally reversible self association function of hnRNPA2 to become irreversible, while the P298L mutation has been proposed to extend an amyloid-prone region by substitution of a key proline with a flexible hydrophobic residue. D290V has been associated with various diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and other neurodegenerative diseases. P298L has been reported only in Paget’s disease of bone. Using a battery of complementary biochemical experiments, the results in this thesis demonstrate that these mutations promote a more stable inclusion body. Specifically, these experiments include quenched hydrogen-deuterium exchange NMR, mass spectrometry, Fourier transform infrared spectroscopy, urea solubilization assays, and limited proteolysis measured by mass spectrometry. Notably, wild-type IB formation appears to depend on the N-terminal affinity tag, whereas both disease variants aggregate independently of it — a result that qualifies direct wild-type-to-mutant comparison and has implications for the use of tagged constructs in aggregation studies. Moreover, D290V gives rise to a generally more stable IB while the effects of P298L present as more subtle and localized in its effects. In conclusion, a wide range of experiments were conducted to characterize IB formation in depth to examine wild-type and disease variant aggregation in a cellular context. Ideally, the work conducted here will help with disease research efforts.