by Caitlin Smith
The inside of a cell is an organized place. Cellular maintenance tasks are functionally compartmentalized into organelles, scaffolding, and appropriately placed proteins, along with lots of other important factors. But how do the proteins know where to go? Though organized into functions, the inside of a cell is also jam-packed. So how does a protein that’s made in the endoplasmic reticulum know to go to the Golgi apparatus for some modifications, then head out to the plasma membrane for awhile, then perhaps visit other organelles when instructed to do so, and then finally end its life in a lysosome? The answer is that we still don’t know; in fact, only about 20% of all proteins have reliably known subcellular locations at all1 (leaving alone the idea for a moment of changing locations). Luckily, new techniques in subcellular protein targeting are bringing in more answers to these intriguing questions.
Predicting subcellular locations
The ability to predict the subcellular home of a protein using statistical analyses of a protein’s sequence can be very useful for scientists studying uncharacterized proteins. The accuracy of these programs is increasing, according to Kuo-Chen Chou, chief scientist and professor at the Gordon Life Science Institute. Chou’s “Cell-PLoc” 2 is a package of web servers containing predictors specialized for eukaryotic, human, plant, Gram-positive bacterial, Gram-negative bacterial and viral proteins. The package incorporates new progress in prediction methods, “reflected by the introduction of the novel concept of ‘pseudo amino acid composition’ ... as well as the introduction of the technique of ‘fusion approach,’” says Chou.
Also improved is the greater number of locations that the newest predictors can cover (up to 22 subcellular locations now compared to 2 to 5 locations in earlier predictors). In addition, the improved ability to predict the locations of multiplex proteins is important because these proteins – which have more than one subcellular location, and may even move between them – can have important physiological functions. But this remains an obstacle, as all predictors today operate on the assumption that each protein has only one location (the one exception being recent work on budding yeast). “The biggest challenge is how we can successfully predict the subcellular locations of the multiplex proteins that can exist in or move among two or more subcellular location sites,” says Chou. According to Chou, about 8% of eukaryotic proteins are reported in the Swiss-Prot database to have multiple subcellular locations. This number could rise, however, with future characterization of proteins that were added in the explosion of work since the Human Genome Project was completed.
Keeping track of multiplex proteins
Two examples of these elusive but crucial multiplex proteins are the glucose transporter 4 (GLUT4) and the P2X receptors for ATP. GLUT4 is sequestered in an intracellular compartment in basal adipocytes. Upon insulin binding to the insulin receptor on the cell surface, GLUT4 transporters traffic to the plasma membrane. GLUT4 is a good example of how some transmembrane proteins carry information about their destinations – via sequence motifs in their cytoplasmic tail(s). GLUT4 has three motifs that are known to be required for proper trafficking, but how exactly they work is not completely resolved. The FQQI and LL motifs are members of families known to be involved with clathrin recruitment and vesicle formation via interactions with adaptor proteins in other systems. The carboxy terminus contains a TELEY motif, but the proteins that bind to these motifs in GLUT4 are unknown. Recent work by the lab of Timothy McGraw, professor of biochemistry at Weill Cornell Medical College, suggests that basal GLUT4 retention in adipocytes requires FQQI and TELEY, but that these motifs regulate different trafficking steps3. Furthermore, the results suggest that the LL motif, along with the AP-1 adaptin complex, are needed to return GLUT4 to the intracellular compartment for retention after the insulin signal has been removed.
P2X receptors are ligand-gated cation channels with two intracellular tails also thought to contain motif-mediated trafficking information. For example, a carboxy terminus motif YXXXK is thought to be involved in keeping the receptor at the plasma membrane. In addition, some subtypes of the P2X receptor may be differentially targeted to lipid rafts in the plasma membrane, where their regulation can be controlled by other signaling molecules that may also be enriched in the rafts. The molecules interacting with the P2X receptors within the rafts are still unknown, but the enrichment of other receptors and signaling molecules in lipid rafts is known for other systems.
Using subcellular targeting for drug delivery
Pharmaceutical researchers are taking advantage of subcellular targeting mechanisms in attempts to deliver therapeutic drugs more effectively. Peter Swaan, professor of pharmaceutical sciences and director of the Center for Nanomedicine and Cellular Delivery at the University of Maryland, studies how to release drugs into a cell via the lysosomal compartment. “Most cargo for subcellular targeting will end up in the lysosome,” he says. “We use specific degradable linkers that target the enzyme Cathepsin B to release drugs within the lysosome. Most small molecules are stable under lysosomal conditions and can escape the lysosomal compartment to enter the cellular cytosol.”
Swaan says that they can target compounds to early endosomes using a variety of plasma membrane receptors that are internalized by clathrin-mediated endocytosis. “By linking a pH-sensitive polymer (containing a drug) to a molecule targeting these receptors, we can effectively enter the cell,” he explains. “Once the endosomal pH lowers due to the recruitment of hydrogen pumps (the classical pathway for this mechanism), the polymers will swell and rupture the endosome, thereby releasing their cargo.” Giving these so-called “nanomedicine complexes” access via the lysosomal pathway could greatly improve our ability to treat lysosomal storage diseases, cancer, and Alzheimer's disease, according to Swaan.
But can we really design a molecule such as an anti-cancer drug that includes information – recognizable by the cell’s inner workings – that could target it directly to its site of action? “It is still difficult to accurately navigate cargo to a specific organelle,” says Swaan, “but the developments and discoveries in cellular biology allow us to get a better understanding of the factors involved in the process of cellular trafficking. Several groups are working on designing chemical tags that would steer a molecule to a specific organelle or compartment.”
One such group is headed by Gus Rosania, assistant professor of pharmaceutical sciences at the University of Michigan. They use high-throughput microscopic imaging to study the distributions and dynamics of small molecules within cells. The data allow them to correlate the structure of small molecules with their subcellular distribution. They suggest that the subcellular distribution of a drug plays a major role in drug efficacy and toxicity. For example, the high intracellular drug concentrations often seen in drug-resistant tumor cells may be explained by drug sequestration in intracellular organelles, rather than drug pumping at the plasma membrane as previously suspected.
Whether by small molecule chemical structure, coupling to compounds, or cytoplasmic sequence motif, proteins and drugs can be targeting to different intracellular compartments with respectable efficiency – but the system of targeting is so complex that there is still much to discover in this interesting era.
References
(1) Chou KC, Shen HB, “Recent progress in protein subcellular location prediction,” Analytical Biochemistry 370: 1–16, 2007.
(2) Chou KC, Shen HB. “Cell-PLoc: a package of web servers for predicting
subcellular localization of proteins in various organisms,” Nature Protocols 3: 153-162, 2008.
(3) Blot V, McGraw TE. “Molecular mechanisms controlling GLUT4 intracellular retention,” Mol Biol Cell 19: 3477-3487, 2008.