The Identification and Targeting of Partially-Folded Conformations on the Folding Free-Energy Landscapes of ALS-Linked Proteins for Therapeutic Intervention: A Dissertation

The hallmark feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the accumulation of cytoplasmic inclusions of key disease-linked proteins. Two of these proteins, TDP-43 and SOD1, represent a significant proportion of sporadic and familial ALS cases, respectively. The population of potentially aggregation-prone partially-folded states on the folding free-energy landscape may serve as a common mechanism for ALS pathogenesis. A detailed biophysical understanding of the folding and misfolding energy landscapes of TDP-43 and SOD1 can provide critical insights into the design of novel therapeutics to delay onset and progression in ALS. Equilibrium unfolding studies on the RNA recognition motif (RRM) domains of TDP-43 revealed the population of a stable RRM intermediate in RRM2, with residual structure localized to the N-terminal half of the domain. Other RRM domains from FUS/TLS and hnRNP A1 similarly populate RRM intermediates, suggesting a possible connection with disease. Mutations, which enhance the population of the RRM2 intermediate, could serve as tools for deciphering the functional and misfolding roles of this partially-folded state in disease models, leading to the development of new biomarkers to track ALS progression. ALS mutations in SOD1 have been shown to destabilize the stable homodimer to result in increased populations of the monomeric and unfolded forms of SOD1. Mechanistic insights into the misfolding of SOD1 demonstrated that the unfolded state is a key species in the initiation and propagation of aggregation, suggesting that limiting these populations may provide therapeutic benefit to ALS patients. An in vitro time-resolved Förster Resonance Energy Transfer assay to screen small molecules that stabilize the native state of SOD1 has identified several lead compounds, providing a pathway to new therapeutics to treat ALS

Folding of the RNA recognition motif (RRM) domains of the amyotrophic lateral sclerosis (ALS)-linked protein TDP-43 reveals an intermediate state

Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that is not observed in RRM1. Strikingly, a portion of RRM2 beginning at position 208, which mimics a cleavage site observed in patient tissues, increases the population of this intermediate state. Mutually stabilizing interactions between the domains in the tethered RRM1 and RRM2 construct reduce the population of the intermediate state and enhance DNA/RNA binding. Despite the high sequence homology of the two domains, a network of large hydrophobic residues in RRM2 provides a possible explanation for the increased stability of RRM2 compared with RRM1. The cluster analysis suggests that the intermediate state may play a functional role by enhancing access to the nuclear export signal contained within its sequence. The intermediate state may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation that may contribute to disease

Occurrence of Euowenia grata (De Vis, 1887) (Diprotodontidae, Marsupialia) from the Pliocene Spring Park Local Fauna, northeastern Queensland

Mackness, B.S., Black, K.H. & Price, G.J., 1.10.2014. Occurrence of Euowenia grata (De Vis, 1887) (Diprotodontidae, Marsupialia) from the Pliocene Spring Park Local Fauna, northeastern Queensland. Alcheringa 39, 000-000. ISSN 0311-551

Possible evidence for intraspecific aggression in a pliocene crocodile from north Queensland

A large, fossilised crocodilian metatarsal has been recovered from the Pliocene Bluff Downs Local Fauna exhibiting proliferative bone growth consistent with an episode of osteoperiostitis, possibly resulting from trauma. The nature and location of this trauma suggests that it may have occurred as a result of intraspecific aggression between rival animals. Three crocodilian genera have been recovered from the Fluff Downs Fauna, Crocodylus, Quinkana and Pallimnarchus. The metatarsal does not conform to Crocodylus porosus and therefore most probably represents one of the other two known crocodilian taxa The nature of the injury suggests that it occurred in water and it may belong to the now extinct Plio-Pleistocene Pallimnarchus rather than Quinkana babarra which is interpreted as being predominantly terrestrial