A series of breakthroughs have been made in the last several months, increasing our knowledge about the structural and functional biology of the VHL protein (pVHL), and helping to advance our knowledge of how pVHL operates in the cell. While there is still a great deal more to learn, some key findings have been published in a series of articles in the journals Science and Molecular and Cellular Biology, and the Proceedings of the National Academy of Sciences. As this issue goes to press, more articles are appearing that we will report in the September issue.

The technology reported in these papers is truly amazing. Unless you too are a molecular biologist, it is an advanced technology completely outside our experience. We are at the dawn of a new era in medicine, so incredibly different from what we have known before that it boggles the mind. In reading these articles and speaking with several of the authors, we have tried to glean the parts of greatest interest to affected families, and to put together some analogies to help us grasp the important messages.

This quest brought to mind Rube Goldberg’s wonderful cartoons of fantastic labor-saving devices at the beginning of the machine age. When Goldberg was born, "the horse and the boat were the principal forms of transportation. There were no automobiles, no superhighways, no airplanes, no radios of television sets, and only a few commercial telephones." Beginning in the 1920’s he hypothesized some modern labor-saving devices, "depicting technology in disarmingly humorous terms. He lived in the midst of the greatest technological revolution that man had ever seen. Electricity and atomic energy were captured and tamed during his eighty-seven years. He lived to see men step on the moon, juggle human hearts from the dead to the living, and send moving pictures through thin air. It was an age of social invention, a time when new machines were being standardized, mass-produced, and marketed for everyone."²

Imagine that you are similarly standing in the 1920’s, looking into the future, at the prospects of all that was ahead. Advances in medical technology, similar to those of machines in the 20th century, await us in the 21st century. Just as Goldberg’s cartoons imagine fanciful and humorous machines (see Figure 1), let us imagine some of the incredible machines that exist in the human body. Picture that we are standing in 1920 with a 1999 radio in our hands. We know that if we turn the knob, music comes out. But how? We take it apart, but we don’t find the simple internals of a music box, instead we find little bits and pieces assembled on a board, with silver lines connecting them. We can see the structure and map it, but we don’t yet understand the function of each of these bits and pieces in bending electrical impulses, taming them to do the job. Little by little we test and deduce the function of each component. Imagine that VHL is one portion of one transistor. From this perspective, let’s take a look at understanding the structure of the VHL protein, map its relationship to the other bits and pieces, and see what is being learned about the probable function of the protein.

Figure 1: Closing the Window

The professor takes a pill and dopes out a device for closing the window if it starts to rain while you’re away.
Pet bullfrog (A), homesick for water, hears rain storm and jumps for joy, pulling string (B), which opens catch (C) and releases hot water bag (D), allowing it to slide under chair (E). Heat raises yeast (F), lifting disk (G) which causes hook (H) to release spring (I). Toy automobile-bumper (J) socks monkey (K) in the neck putting him down for the count on table (L). He staggers to his feet and slips on banana peel (M). He instinctively reaches for flying rings (N) to avoid further disaster and his weight pulls rope (O) closing window (P), stopping the rain from leaking through on the family downstairs and thinning their soup.

RUBE GOLDBERG™ and copyright of Rube Goldberg Inc., Distributed by United Media. Reprinted with permission.

Why is this important? It is important so that we can learn what happens when the VHL protein is present and fully functional, and what goes on when the VHL protein is missing or not quite right. It is important for us to find out what we can do to intervene at some point along the chain and make a difference in the outcome. If, for example, in Goldberg’s machine (Figure 1) the bread dough (E) doesn’t rise as high as expected, and the monkey continues to snooze, none of the actions G through P will occur. Might there be some other way to wake up the monkey? Or if we simply do something entirely different to entice the monkey to grab the rings (N) we would still get the same result. Where else could we intervene and make a difference in the outcome?

The VHL gene encodes a protein essential to the normal functioning of the cell. A protein is a long string of amino acids that folds into a 3-dimensional structure with a specific shape, depicted as a ribbon diagram (see Figure 2). The shape is important to its ability to connect with other proteins and enzymes and therefore for its function.

When there is a change in one of the amino acids in the chain, one of two things may happen: (1) the change may cause it to unravel, or (2) other changes on the surface of the protein may interfere with its ability to form at least one important connection. Picture a set of electronic components that have to fit together in a specific way in order for the larger complex to do its larger job. If the coupler is damaged, or one of the pins on the transistor is broken off, the two parts don’t fit together properly.

The shape of the VHL protein is published for the first time this month in Science.³ (See Figure 2.) In addition, two other proteins that bind closely with VHL are identified and their shapes described as well. The ribbon diagram shows the physical shape of these proteins, and the way they fit together. A "binding region" has been identified along the VHL gene, which serves as the connection point where ElonginC binds to VHL. ElonginC in turn binds to ElonginB, forming a three-part VCB complex. A second area along the VHL gene forms a second binding site. The current hypothesis is that this second site is used to attach one of several proteins to the VCB complex.

Figure 2: Structure of VHL Protein

The VCB 3-part complex consists of two interfaces, one between VHL and ElonginC and the other between ElonginC and ElonginB. This ribbon diagram illustrates the secondary structure of the VCB complex. The H4 helix of ElonginC, which bulges out from the side of the concave surface, fits into an extended groove formed by the H1, H2, and H3 helices of the VHL alpha domain. The H1 helix of the VHL alpha domain fits into the concave surface of ElonginC and is important for ElonginC binding. About half of VHL missense mutations occur in these critical binding areas. VHL Type II mutations (with pheochromocytoma) frequently occur in the ElonginC binding area or the secondary protein-binding site. VHL Type I mutations frequently occur in the beta domain core, causing a more complete unraveling of the VHL structure. Thus Type II mutations more often cause partial loss of function. (C marks the COOH terminus (the end); N marks the NH2-terminus (the beginning of the numbering sequence).

Stebbins et al, Science, 284:455. Reprinted with permission of the author.

click here for color

Interestingly, when we look at the locations of the point mutations found in people who have the condition we call VHL, we find that those mutations generally occur at these binding sites. Therefore the hypothesis is that when the binding site is disrupted and the connection with Elongins C and B cannot be made correctly, a change occurs in the cell’s life cycle.

"The structure of VHL allows sense to be made of the rich database of known VHL mutations in tumors."⁴ One group of mutations clusters in an area where they probably disrupt binding to ElonginC, whereas another group clusters in an area which appears to define a binding surface for an as yet unidentified protein, suggesting that VHL serves as a kind of adapter, attaching one or more proteins on one end to the Elongins on the other end. This structure and function looks very similar to another complex, the SCF⁵ complex, which seems to play a role in regulating the levels of certain cellular proteins in the cell by targeting them for destruction, a process called proteolysis. There is reason to believe that VHL plays a similar regulatory role, taking certain cellular proteins from the cytoplasm of the cell and targeting them for destruction.

Scientists also reported discovery of a new protein, Rbx1, that is a part of the VHL tumor suppressor complex or machinery. Remarkably, Rbx1 is also found as a critical component of the machinery that controls cell growth and division by targeting important regulatory proteins for destruction.

"An exciting hypothesis is that, in its normal form, VHL works together with Rbx1 to prevent the cell from accumulating proteins that trigger uncontrolled cell proliferation. But a mutation in the gene that produces VHL can cause cancer by interfering with the cell’s ability to destroy the trigger proteins, leading to runaway cell growth," said Joan Conaway, Howard Hughes Medical Institute investigator at OMRF.

Notes Ron Conaway, "Not surprisingly, the mechanisms that regulate protein destruction are turning out to be every bit as complicated and every bit as important as those that regulate protein synthesis. The discovery of the link between VHL, Rbx1, and the protein destruction machinery should help researchers identify the specific trigger proteins targeted by VHL and may lead to new approaches to cancer prevention and treatment."

In 1993 the Conaways discovered and isolated the Elongins as proteins that help to control the mechanism that turns genes, including cancer genes, "on" or "off." In 1995 they collaborated with Drs. Richard Klausner and Marston Linehan of the National Institutes of Health (NIH) to demonstrate that the Elongins function in cells with the VHL tumor suppressor protein to prevent cancer.

"These findings represent a significant advance in our understanding of the function of the VHL gene and how damage to this gene leads to the manifestations in patients that we know of as cancer," said W. Marston Linehan, M.D., Chief of the Urologic Oncology Branch at the National Cancer Institute. "We strongly feel that it is work such as this that will one day play a major role in the development of effective forms of therapy for patients with kidney cancer."

What's Next? How Can We Help?

Based on these new understandings, additional research teams will form new conjectures about the effects of VHL on various cell functions, and will set out to prove or disprove them in the lab. This will lead to new therapies for VHL and other cancers.

The VHL families can assist by continuing to donate tumor tissue to the VHL Tissue Bank⁶, and by participating in research studies whenever they are appropriate. It takes all of us -- families, clinicians, research scientists, and donors of research money -- to make the kind of progress we are seeing. It also takes work to ensure that society uses this new information responsibly for good, not as a new tool for discrimination. We look forward to the scale of advances in genetics and medicine that Rube Goldberg saw in machines in his lifetime. The human body is the ultimate incredible machine.

To Register for the Tissue Bank, click here.

1. Stebbins et al, "Structure of the VHL-ElinginC-ElonginB Complex: Implications for VHL Tumor Suppressor Function," Science, 284:455, 16 April 1999; Tyers and Willems, "One Ring to Rule a Superfamily of E3 Ubiquitin Ligases," Science, 284:601, 23 April 1999; Skowyra, Conaway, et al., "Reconstitution of G1 Cyclin Ubiquitination with Complexes Containing SCFGrr1 and Rbx1," Science, 284:662, 23 April 1999; Kamura, Iliopoulos, Kaelin, Conaway et al, "Rbx1, a Component of the VHL Tumor Suppressor Complex and SCF Ubiquitin Ligase," Science 284:657, 23 April 1999; Lee, Pause, Klausner et al, "Transcription-Dependent Nuclear-Cytoplasmic Trafficking Is Required for the Function of the von Hippel-Lindau Tumor Suppressor Protein," Mol. Cell. Biol. 19:1486, 1999; Gorospe, Zbar, Lerman et al, "Protective Function of von Hippel-Lindau Protein against Impaired Protein Processing in Renal Carcinoma Cells," Mol. Cell. Biol. 19:1289, 1999; Liakopoulos, Pause, et al., "Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel-Lindau tumor suppressor function", PNAS, May 1999. Our thanks to Myriam Gorospe, Charles Stebbins, Othon Iliopoulos, and Joan and Ron Conaway for their assistance in preparing this article.

2. Marzio, Goldberg, p. 145.
3. C. Stebbins, W. Kaelin, and N. Pavletich, "Structure", Science 284:455.
4. Tyers and Willems, "Ring," Science, 284:601
5. Skp1-Cdc53/CUL1-F-box protein. Another similar structure, APC, has also been identified as a possible ubiquitin ligase complex. It is believed that each of these three complexes mediate the targeted degradation of many cellular proteins. Targetting proteins for degradation requires ubiquitin, a small protein that can be attached to other proteins, thereby ‘marking’ them for deletion, in much the same way that a forester marks trees to be cut down. Skowyra et al., provide a relevant example of the importance of this protein degradation pathway (Science 284, 23). Cyclins are proteins that trigger cell division, and their elimination was shown to be mediated by the SCF complex, which is very homologous to the VHL complex. If the degradation complex (SCF or VHL) is disrupted due to mutations or due to the absence of certain complex components, these cyclins cannot be eliminated, and the cell division cycle continues uncontrolled, as in tumors. Therefore, protein destruction is a very important way of ‘keeping in check’ cellular growth and keeping tissues and organs healthy.

6. The VHL Tissue Bank keeps surgically removed tumor tissue on file so that researchers will have the cells they need to study. As the level of VHL research increases, the need for VHL tissue for research also increases. It is here that we can help. If you have been diagnosed with VHL, and are contemplating surgery, you can help the research community by donating any surgically removed tissue to the VHL Tissue Bank. All cost and arrangements for recovery and transfer of tissue will be taken care of by the Tissue Bank. If you would like to help the VHL research effort, please fill out the Donor Registration Form and mail it today. All information will be treated in the strictest confidence. Pre-registration makes the process simple in the event of surgery. Simply contact the tissue bank, give them the name and contact information for the surgeon and the date of surgery, and the Tissue Bank will make all the necessary arrangements. Even if you are not already pre-registered, arrangements can be made by contacting the Tissue Bank. Outside the U.S., please call the nearest Affiliate for similar programs near you.

As printed in the VHL Family Forum 7:2, June 1999. For permission to reprint, please contact VHL Family Alliance, info@vhl.org. Further information is available from the VHL Family Alliance, info@vhl.org.

research