Mechanisms for getting stuff into and out of cells are of great importance. A new item of research came to my attention related to substance-trafficking that goes on within cells and across cell membrane barriers. It has relevancy in terms of several disease processes and aging. I review the research in the context of the fascinating cell biology involved.
We humans may have around 100 trillion eukaryotic cells, complex entities consisting of many components and constantly engaged in many internal activities. Some of these activities involve transportation of substances either within cells or from the cell’s outer membrane surface into the cell or visa-a-versa. For those of you not familiar with cell biology I need to identify a number of cell entities that are important for understanding how substance-trafficking in cells works. I will try to keep things as simple as possible for the purpose of this discussion. Vesicles are little bubbles of fluid in cells surrounded by lipid bilayers that serve a number of functions including molecular cargo transportation. Among other things, they can transport needed substances into a cell or unwanted ones out. “Vesicles store, transport, or digest cellular products and waste. The membrane enclosing the vesicle is similar to that of the plasma membrane, and vesicles can fuse with the plasma membrane to release their contents outside of the cell. Vesicles can also fuse with other organelles within the cell.” “ — an endosome is a membrane bound compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation, or they can be recycled back to the plasma membrane. Molecules are also transported to endosomes from the Golgi and either continue to lysosomes or recycle back to the Golgi. — Early Endosomes Consist of a dynamic tubular-vesicular network (vesicles up to 1 µm in diameter with connected tubules of approx. 50 nm diameter). Markers include RAB5 and RAB4, Transferrin and its receptor and EEA1(ref).“ “Lysosomes are spherical organelles that contain enzymes (acid hydrolases). They break up food so it is easier to digest. — Lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria(ref).” Finally, “the Gogli apparatus (also Golgi body) is an organelle found in most eukaryotic cells. The primary function of the Golgi apparatus is to process and package macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination; it is particularly important in the processing of proteins for secretion. The Golgi apparatus forms a part of the cellular endomembrane system(ref).” OK, enough biological distinctions for now.
The new research
The February 2010 paper The Connecdenn DENN Domain: A GEF for Rab35 Mediating Cargo-Specific Exit from Early Endosomes deals with signaling that tells vesicles how fast they have to move when they are transporting substances to and from the surfaces of cells. According to ScienceDaily (Mar. 18, 2010) “Defects in this trafficking pathway can have severe consequences, leading to numerous diseases such as high cholesterol, neuropathies, sterility and complications in immune response.– Dr. Peter McPherson and Dr. Brigitte Ritter and their colleagues have discovered how a molecule called Rab35, which acts as a switch is turned on in order to activate the fast-track recycling pathway — in which cargo that needs to be recycled back to the surface of the cell is rapidly selected and transported. — “The cells that make up our bodies are like a busy city,” says Dr. McPherson, neuroscientist at The Neuro and the co-principal investigator for the study. “The cell surface is defined by a membrane that separates its interior from the external world, like the walls or borders of a city. Within this environment, there are simultaneous trafficking pathways that transport vital nutrients, receptors and other components required for cells to function, within cargo vehicles called ‘vesicles.’ Like traffic in a city, these ‘cargo’ vesicles travel at different speeds to numerous destinations within the cell with different purposes. For example, the receptors on the cell surface that bind to cholesterol are on the fast track pathway, so that once they deliver the cholesterol inside the cell, they move back to the surface quickly so that they can pick up some more. It is therefore crucial to understand the controls and switching mechanisms of trafficking inside cells, as this system is of vital importance to the proper functioning of the body.”
The ScienceDaily article continues “The Rab35 molecule is the trafficking switch for the fast-track or high-priority recycling pathway signaling the quick return of cargo to the cell surface membrane. It is known that Rab35 exists in two forms, ‘on’ (GTP- bound) or off (GDP- bound). When Rab35 is turned ‘on’, it allows the cargo to go back up to the cell surface. What Dr. McPherson and Dr. Ritter and colleagues have discovered is the switch that turns Rab35 on. — “In this study we identified that a particular region of the vesicle-bound protein connecden, called the DENN domain, is the ‘finger’ that flips the switch,” says Dr. Ritter. “The DENN domain connects with the Rab35 molecule and through enzymatic activity converts Rab35 from the inactive to the active form, in essence, turning on the switch.” You can find articles relevant to the DENN domain here. According to the new publication the DENN domain is a lipid-binding module with enzymatic guanine nucleotide exchange factor (GEF) activity for Rab35, and Rab35 controls cargo-specific recycling from early endosomes. “The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.”
A second February 2010 relevant e-publication ahead of print is The connecdenn family: Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery. ‘We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35 whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C-termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2.” Clathrin is a protein which plays a major role in the formation of coated vesicles.
The ScienceDaily article points to the importance of the new results in helping us understand healthy cell cargo transportation: “If the finger or the switch itself is mutated or missing, cargo can’t recycle, which has dire consequences,” adds Dr. McPherson. “For example a very important cargo transported by this specific fast track recycling pathway, controlled by Rab35 is the MHC class I receptor involved in the immune system response. If a cell becomes infected by a virus, the MHC receptor is loaded with fragments of the virus that have infected the inside of a cell. The MHC receptor needs to be taken back to the cell surface quickly so that so that it can act as a signpost indicating to circulating immune cells that this particular cell has been infected by a virus and needs to be destroyed, preventing viral infection to other cells.” If the cargo trafficking system can’t work well the immune system won’t be able to do its job well and the result will be disease and premature aging.
So, some diseases and aging processes are consequent to problems associated with cell substance-trafficking. New insights being developed related to the control mechanisms for the cells’ trafficking system are likely to prove very useful.
Nanoparticles for curing cancer.
“What’s so exciting is that virtually any gene can be targeted now,” he says. “Every protein now is druggable.”
That research indeed seems exciting to me though larger-scale trials are clearly needed. The area of RNAi has been one of great theoretical interest. Now at last, using nanoparticles perhaps RNAi can be put to large-scale therapeutic use. I have been generally skeptical about predictions of use of nanotechnology to support longevity (e.g. nanomachines that go about like tiny surgeons repairing organs). However I think that this application, marrying nanotechnology to molecular biology, indeed might have a chance of being a big winner.