Kopito Lab Projects

Research projects in the lab are focused on the following questions:

• How are misfolded proteins recognized and degraded?
• What are protein aggregates and inclusion bodies?
• What is the relationship between protein aggregation and neurodegenerative diseases such as Alzheimer, Parkinson, Lou Gehrig, etc.?

How are misfolded proteins recognized and degraded?

The synthesis of integral membrane and secretory proteins begins with cotranslational translocation of the nascent polypeptide into the membrane of the endoplasmic reticulum (ER). In 1995, our lab, together with several other groups decovered that misfolded integral membrane proteins can be retro-translocated (‘dislocated’) across the ER membrane by which these misfolded proteins are recognized and delivered to the proteasome.

We’re currently utilizing two model systems to investigate the degradation of misfolded proteins that are translocated into the ER.

Model system #1 (Pic)

TCR a. The T-cell antigen receptor is a heterooligomeric complex of 7-9 polypeptide chains, all of which are type I integral membrane proteins. Assembly of the complex is inefficient; excess a-chains are degraded by cytoplasmic proteasomes.

Model system #2 (Pic)

CFTR. Cystic fibrosis (CF) is the most common lethal inherited disorder among Caucasians. It is caused by mutations in a gene on chromosome 7 that encodes that cystic fibrosis transmembrane conductance regulator (CFTR) , a polytopic integral membrane glycoprotein related to the ABC family of membrane pumps. The vast majority of US and European CF patients have inherited at least one copy of the DF508 allele, which deletes a single amino acid from a cytoplasmic domain of the protein. DF508 CFTR molecules are synthesized normally, but are unable to fold. We have shown that they are rapidly dislocated from the ER and degraded by cytoplasmic proteasomes.

Protein aggregates and Inclusion bodies.

In cells, aggregated proteins are usually found in inclusion bodies (Pic), structures that contain extremely high concentrations of aggregated proteins. Inclusion bodies form in cells or organelles when they are forced to express heterologous or mutant proteins. Inclusion bodies can also form upon overexpression of some endogenous proteins, suggesting that the machinery for folding and/or processing can be saturated.

Aggregation. ‘Conventional wisdom’ is that protein aggregates are non-specific junk piles of misfolded, jumbled proteins, sort of like molecular dustballs, or scrambled eggs. Our data suggest that protein aggregates are alternative conformational states of proteins and that their formation is no less specific than formation of the native state. We are using fluorescence resonance energy transfer (FRET), electron microscopy, and biochemical techniques to study the formation of protein aggregates in vivo.

What is the relationship between protein aggregation and neurodegenerative diseases?

Nearly all neurodegenerative diseases (and many other human diseases as wells) are tightly linked to cellular pathology including the formation of intracellular inclusion bodies, the accumulatiohn of ubiquitin conjugates and cytoskeletal abnormalities. There is stron genetic evidence supporting the hypothesis that neuron death in several neurodegenerative diseases may be a direct consequence of protein aggregation.

How can aggregates be toxic to cells?

1. Do these diseases reflect a failure of the cellular quality control machinery to suppress protein misfolding and aggregation?

Our lab is investigating these questions in several model systems:

Polyglutamine Diseases (Huntington’s, Ataxins, Kennedy’s Disease, etc.) & Amyotrophic Lateral Sclerosis.