FRAP and recovery of GFPSec61 g fluorescence into Organized Smooth ER whorl.The focus of our laboratory is to understand the cell biology of secretory protein synthesis. Specifically, we are using a combination of live cell imaging, biophysical fluorescence methods, biochemistry, and molecular biology to study the regulation, organization and dynamics of secretory protein translocation and folding in cells. Cutting edge quantitative fluorescence microscopy methods including FRAP, FLIP, photoactivation and FRET reveal information about protein mobility, environment, protein complex size, protein-protein interactions, and membrane dynamics in living cells.
Confocal micrograph of a Cos-7 cell expressing a lumenal ER GFPThe endoplasmic reticulum (ER) is the largest eukaryotic organelle and carries out multiple functions including:
1) secretory protein bio-synthesis, and export
2) protein glycosylation and disulfide bond formation
To prevent protein misfolding, the ER contains a variety of proteins, termed chaperones. A functional secretory protein must be successfully translocated into the ER and then interact with the correct subset of chaperones to correctly fold. Failure of any one of these steps can lead to protein-misfolding diseases, such as cystic fibrosis. At the same time, regulation of these processes can be exploited by viruses, such as HIV, and appears to be important in genetic diseases such as polycystic liver disease.
Regulation, organization, dynamics of ER translocation and quality control machinery during aging
Analysis of the human genome has revealed that one fourth of all proteins are either membrane or secretory proteins. These proteins enter the ER through a large multi-protein channel termed the translocon. As the nascent peptides co-translationally insert into the translocon, the peptides encounter chaperones, which promote proper folding, disulfide bond formation, prevent protein aggregation, and form the basis of ER quality control. Some chaperones appear to assemble into multi-protein complexes. We are interested in the defining the composition and dynamics of these complexes in cells.
Misfolded transmembrane and secretory proteins frequently accumulate in aging cells and are a feature of several age-associated diseases, such as Alzheimer’s Disease and inclusion body myositis. Oxidative damage of some chaperones in aging tissues suggests that dysfunction of the protein folding machinery contributes to age-related protein misfolding.
In this project, we are investigating the role of oxidative damage of protein folding components in age-related protein misfolding using two approaches: 1) We are biochemically characterizing age-related changes in levels of chaperone and translocon components and the extent of oxidative damage to these components in aging mouse liver. 2) Using cultured cells, we assess the function, organization, dynamics, and stability of oxidatively-damaged chaperone and translocon components both biochemically and with novel imaging techniques. Understanding the role of oxidative damage in protein misfolding will lead to new strategies for preventing protein folding diseases of aging.
Secretory Protein Translocation Efficiency
Secretory protein translocation and folding Secretory proteins are targeted to the ER translocon with a signal sequence- a protein domain of 14-50 amino acids typically located at the NH2-terminus of the protein. Surprisingly, every secretory protein has a different signal sequence. The signal sequence is usually cleaved from the protein as the protein is translocated into the ER. Different cells appear to translocate different signal sequences with differing efficiencies. We are interested in the consequences of this phenomenon.
Recent studies suggest that inefficient translocation is important for disease and for previously unappreciated roles of proteins traditionally considered ER proteins- such as the lumenal chaperone calreticulin, which also appears to have functional roles in the cytoplasm. To better understand the roles of signal sequences, we are characterizing "inefficient signals" and signal sequence associated diseases.
Interaction between the signal sequence and translocon modulates protein folding and localization:
Outcomes A, C, and E represent folded functional forms of the same protein that result due to differences in signal sequence translocation efficiency. B. An incorrectly folded protein. D. Most secretory proteins that fail to translocate are degraded in the cytoplasm.
Secretory Protein Processing in HIV
The HIV-1 virus has an envelope glycoprotein gp160 that has significant protein folding requirements including 30 glycosylation sites and 10 disulfides. The signal sequence of gp160 appears to be removed minutes to hours after the protein has been synthesized and translocated. We are studying the regulation of gp160 signal seuqence processing. We hypothesize that this is important for viral production and pathogenicity.
Fluorescent Protein Resource Center
Dr. Snapp is the co-director of the Einstein Fluorescent Protein Resource Center. For information on the latest fluorescent proteins and their uses for live cell imaging, please go to the Resource Center website: www.einsteinmed.org/research/facilities/fluorescent/
The Snapp lab is taking rotation students and is seeking a postdoctoral fellow. Please contact Dr. Snapp to schedule an interview.
Erik Lee Snapp is an Ellison Medical Foundation New Scholar in Aging and a recipient of pilot awards from Einstein's CFAR, Einstein's Marion Bessin Liver Center, and Einstein's Resnick Gerontology Center.
