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The Rochet Lab
Protein Misassembly Diseases

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Research in my laboratory is aimed at understanding the role of protein aggregation in neurological and muscular disorders. Generally, proteins adopt a precise three-dimensional structure that determines their function. However, mutant polypeptides or proteins subjected to environmental stress are often incorrectly folded. At high concentrations, proteins that lack a compact fold tend to undergo spurious interactions to form abnormal, high-molecular-weight complexes (Figure 1). This phenomenon, referred to as protein 'aggregation' or 'misassembly', is associated with various human diseases. We have chosen to address the role of protein misassembly in three of these disorders: Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and desmin-related myopathy (DRM). Importantly, heritable forms of each disorder have been linked to the gene that encodes the characteristic misassembled protein associated with the disease. This observation is taken as evidence that protein aggregation plays a role in the pathogenesis of these diseases.

Figure 1

Figure 1: Diagram illustrating the formation of an ordered aggregate (β-sheet-rich fibril) from a partially unfolded protein

Parkinson's Disease

PD is a neurodegenerative disorder characterized by the loss of dopaminergic neurons from the substantia nigra of the brain. Some surviving nigral neurons contain intracellular inclusions (Lewy bodies) that are enriched with fibrils of the presynaptic protein a-synuclein. Rare, early-onset, familial forms of PD have been linked to mutations in the gene encoding a-synuclein. The results of studies using purified wild-type (WT) and mutant a-synuclein have led to the hypothesis that a-synuclein protofibrils rather than fibrils are toxic to dopaminergic neurons in PD. Protofibrillar but not fibrillar a-synuclein permeabilizes synthetic vesicles, suggesting that the protofibrils selectively cause neurodegeneration via membrane disruption (Figure 2). Dopamine reacts with a-synuclein to form a covalent adduct, which in turn stabilizes protofibrils and inhibits their conversion to mature fibrils.

Figure 2

Figure 2: Model illustrating how ?-synuclein protofibrils may be toxic in PD

Amyotrophic Lateral Sclerosis

ALS is a neurodegenerative disorder caused by the loss of upper and lower motor neurons. Protein aggregates characteristic of this disease contain superoxide dismutase (SOD1) and neurofilaments. Approximately 20% of familial ALS cases are due to mutations in the gene encoding SOD1, a cytosolic enzyme that converts superoxide to water and hydrogen peroxide. The link between mutant SOD1 and familial ALS may result from the enhanced propensity of the mutant enzyme to form toxic aggregates compared to WT SOD1 (Figure 3). Alternatively, mutant SOD1 may catalyze aberrant reactions that lead to the post-translational modification and ensuing aggregation of neurofilaments. Neurofilaments are intermediate filaments that consist of heavy (H), medium (M), and light (L) subunits and are essential for maintaining the structural integrity of the cell and regulating axonal transport. Neurofilament (NF)-containing inclusions may cause cell death in ALS via axonal strangulation. Consistent with this hypothesis, mutations in the gene encoding NF-H have been linked to ALS.

Figure 3

Figure 3: Two models to explain the role of SOD1- and NF-misassembly in ALS

Desmin-Related Myopathy

The term 'desmin-related myopathy' encompasses a heterogeneous group of skeletal and cardiac myopathies. The muscle tissue of DRM is characterized by split or misaligned myofibrils and inclusions that contain the intermediate filament protein desmin. Several cases of familial DRM have been linked to mutations in the gene encoding desmin. Another case of familial DRM has been linked to a mutation in the gene encoding αβ-crystallin, a small heat-shock protein that functions as a chaperone to prevent the misassembly of desmin. Mutant αβ-crystallin may cause the aggregation of desmin via a toxic gain of function (for example, by promoting the hyperphosphorylation of desmin) (Figure 4).

Figure 4

Figure 4: Interactions between ?B-crystallin and desmin

For all three diseases, we aim to answer the following questions:

  1. What are the molecular mechanisms (intermolecular interactions, conformational changes) underlying protein misassembly?
  2. How do post-translational modifications (e.g. oxidation, phosphorylation, ubiquitylation) affect protein aggregation?
  3. What are the cellular factors (e.g. chaperones, ubiquitin-conjugating enzymes, proteasome) that modulate protein misassembly?
  4. What are the molecular pathways (e.g. apoptosis, necrosis) by which protein aggregation causes disease?

These questions are addressed using an interdisciplinary approach, including biochemical and biophysical analyses of recombinant proteins and the development of yeast (Figure 5) and mammalian cell models. Studies will also be carried out using transgenic Drosophila and C. elegans. The advantage of transgenic flies and worms as disease models is that they can be analyzed using reverse-genetic approaches to decipher molecular pathways underlying pathogenesis.

Figure 5

Figure 5: S. cerevisiae engineered to produce α-synuclein in one of two states: aggregated (A, C) or diffuse (B, D)

Finally, an important goal is to identify small molecules via high-throughput screening to study and ultimately treat these devastating illnesses.


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This page last updated Thursday, April 20, 2006 at 15:43.