Case Presentation

1. Case Presentation 49 year old white female who presents for evaluation of liver tumors She has a history of retinal angiomas that developed when she was 13 years old. These have been treated with multiple laser surgeries and she is now blind on the left. She also has a history of an ACTH-producing tumor in the lung that was resected in 1996.

2. Case continued ACTH producing neuroendocrine tumors were also found in the liver. These were treated with resection in 1998 and then radiofrequency ablation in 2001 for recurrent symptomatic lesions.

3. Case continued Also history of renal cell carcinomas. These have been resected in 1996 then in 2001. Other history includes a history of BAD, and surgeries after trauma. Medications include synthroid, psychiatric drugs, darvocet, celebrex.

4. Case continued Family history is significant for a father who died of heart disease at 81 and a mother who died of dementia at 73. She has 8 healthy sibs. She has one child 25 y/o with spina bifida, one with IDDM at 14 y/o, and a 20 y/o with learning disability. She lives with her husband and two children. She has 30 pack year of tobacco and quit 3 years ago. She drinks occasional alcohol and works in the home now.

5. Physical exam Obese white female with striae over her abdomen and axillae. P=92 BP=137/83 Wt 183 lb Afebrile Surgical scars on right thorax and on the abdomen across the midline to the right. Pupils with surgical changes bilaterally, left eye deviated laterally. Otherwise, unremarkable.

6. Radiographic Data CT of chest, abdomen, and pelvis remarkable for several arterial enhancing liver lesions. Also there is a pancreatic cyst and a mass consistent with a microcystic cystadenoma. Kidneys have multiple bilateral renal cysts.

9. Von Hippel-Lindau Syndrome Autosomal dominant tumor with multiple benign and malignant tumors in multiple organ systems. Hallmarks include retinal angiomas, cerebellar and spinal hemangioblastomas, and renal cell carcinomas. Also see cystic lesions in the kidneys, pancreas and epididymitis. Adrenal pheochromocytomas occur in 7-20% of those affected. The presence of pheos defines the disease as Type 2.

10. VHL Prevalence is around 1/40,000 in Western European countries. Affects sexes equally and is seen in all ethnic groups. Penetrance is estimated at 80-90% by 65 years old. Diagnosed at infancy thru seventh decade.

12. Diagnostic Criteria Single retinal or cerebellar hemangioblastom,RCC, or pheo is sufficient in someone with a known family history. In an isolated case, diagnosis is made with 2 or more hemangioblastomas or a single hemangioblastoma and a characteristic visceral tumor. Type I--No pheo Type 2A pheo without RCC and 2B with both.

13. Molecular Basis Positional cloning in families with overlapping germline deletions led to the identification of the VHL gene on 3p25-26. VHL is a tumor suppressor gene that follows Knudson�s 2-hit hypothesis. In VHL families, the wild-type allele was lost in VHL tumors.

14. VHL gene product 213 amino acids with a molecular mass of the protein of approx. 20-30kDa mRNA expressed in all adult tissues. Mutations are heterogeneous. 15-20% have large deletions. 27% with missense and 27% with framshift or nonsense mutations. Mutations identified in nearly all VHL families. In Type 2- 96% have missense whereas deletion and premature termination is associated with I. Ubiquitous expression means that this does not explain tumor types.Ubiquitous expression means that this does not explain tumor types.

15. VHL protein Binds Elongin B and C. Elongin B and C are components of a transcriptional elongation complex called Elongin/SIII It was initially thought that this explained the mechanism of action of VHLp. Namely nl VHL prevented elongin A from binding and therefore prevented transcription of certain genes. but yeast homology and the excess of elongin B and C cell suggested otherwise.It was initially thought that this explained the mechanism of action of VHLp. Namely nl VHL prevented elongin A from binding and therefore prevented transcription of certain genes. but yeast homology and the excess of elongin B and C cell suggested otherwise.

16. pVHL and Ubiquitination pVHL forms stable complexes with the elongins and Cul2 and Rbx1. Cul2 and elongin C showed homology to yeast E3 ubiquitin ligases that were involved in substrate recognition for ubiquitination. Is pVHL involved in ubiquitination?

17. Ubiquitination Review(Simplified) Ubiquitin uses ATP to form a thiol ester intermediate with E1-ubiquitin activating enzyme. Activated ubiquitin is then transferred to E2 from E1. In the presence of E3, E2 transfers ubiquitin to the specific substrate. Polyubiquitinated proteins are degraded by the 26S proteasome. U forms a isopeptide bond between the C-terminal glycine abd ab E-amino group of a lysine residue of the substrate protein.U forms a isopeptide bond between the C-terminal glycine abd ab E-amino group of a lysine residue of the substrate protein.

18. Fig. 1. VBC-CUL-2 exhibits the E3 activity together with E2s of UbcH5 family. (A) The VBC-CUL-2 complex immunoprecipitated from HA-tagged WT-pVHL expressing 786-0 cells was incubated with recombinant E1, 32P-labeled ubiquitin, and ATP regeneration system at 37�C for 30 min in the presence of UBC3 (lane 2), UbcH5a (lane 3), UbcH5b (lane 4), UbcH5c (lane 5), UbcH7 (lane 6), E2-25K (lane 7), or E2-20K (lane 8), or in the absence of any E2 (lane 1). (B) In vitro ubiquitination reactions were performed as described in Materials and Methods, except for the followings: lane 1 lacks VBC-CUL-2, lane 3 lacks ATP regeneration system, lane 4 lacks UbcH5b, and lane 5 lacks E1. (C) Immunoprecipitates with anti-HA agarose beads from HA-tagged WT-pVHL expressing (lane 3), HA-tagged truncated pVHL (amino acids 1-115) expressing (lane 4), or parent 786-0 cells (lane 2) were subjected to the in vitro ubiquitination assay together with anti-HA beads alone (lane 1) in the presence of E1, UbcH5b, 32P-labeled ubiquitin, and ATP regeneration system at 37�C for 30 min. Reactions were stopped by adding 4? sample buffer and electrophoresed in 10% SDS/PAGE, followed by autoradiography. Fig. 1. VBC-CUL-2 exhibits the E3 activity together with E2s of UbcH5 family. (A) The VBC-CUL-2 complex immunoprecipitated from HA-tagged WT-pVHL expressing 786-0 cells was incubated with recombinant E1, 32P-labeled ubiquitin, and ATP regeneration system at 37�C for 30 min in the presence of UBC3 (lane 2), UbcH5a (lane 3), UbcH5b (lane 4), UbcH5c (lane 5), UbcH7 (lane 6), E2-25K (lane 7), or E2-20K (lane 8), or in the absence of any E2 (lane 1). (B) In vitro ubiquitination reactions were performed as described in Materials and Methods, except for the followings: lane 1 lacks VBC-CUL-2, lane 3 lacks ATP regeneration system, lane 4 lacks UbcH5b, and lane 5 lacks E1. (C) Immunoprecipitates with anti-HA agarose beads from HA-tagged WT-pVHL expressing (lane 3), HA-tagged truncated pVHL (amino acids 1-115) expressing (lane 4), or parent 786-0 cells (lane 2) were subjected to the in vitro ubiquitination assay together with anti-HA beads alone (lane 1) in the presence of E1, UbcH5b, 32P-labeled ubiquitin, and ATP regeneration system at 37�C for 30 min. Reactions were stopped by adding 4? sample buffer and electrophoresed in 10% SDS/PAGE, followed by autoradiography.

19. Fig. 4. VHL mutants found in patients failed to ubiquitinate p220 and p100. Hi Five cells infected with recombinant baculovirus encoding CUL-2, elongin B, elongin C, and Rbx1 (lane 2) together with WT-pVHL (lane 3), P86H (lane 4), Y98N (lane 5), or L158P (lane 6) encoding recombinant baculoviruses. Lysates of infected cells or noninfected cells (lane 1) were immunoprecipitated with anti-Flag M2 beads. (A) Immunoprecipitates were subjected to in vitro ubiquitination assay as described in Fig. 1C. Samples were electrophoresed in 9% SDS/PAGE, followed by autoradiography. (B) Immunoprecipitates were electrophoresed in 4-20% gradient SDS/PAGE. After being transferred, the membrane was probed with anti-HA, anti-Flag M2, anti-HSV, or anti-T7 antibodies, to detect CUL-2, pVHL, elongin C (Elo C), or Rbx1, respectively.Fig. 4. VHL mutants found in patients failed to ubiquitinate p220 and p100. Hi Five cells infected with recombinant baculovirus encoding CUL-2, elongin B, elongin C, and Rbx1 (lane 2) together with WT-pVHL (lane 3), P86H (lane 4), Y98N (lane 5), or L158P (lane 6) encoding recombinant baculoviruses. Lysates of infected cells or noninfected cells (lane 1) were immunoprecipitated with anti-Flag M2 beads. (A) Immunoprecipitates were subjected to in vitro ubiquitination assay as described in Fig. 1C. Samples were electrophoresed in 9% SDS/PAGE, followed by autoradiography. (B) Immunoprecipitates were electrophoresed in 4-20% gradient SDS/PAGE. After being transferred, the membrane was probed with anti-HA, anti-Flag M2, anti-HSV, or anti-T7 antibodies, to detect CUL-2, pVHL, elongin C (Elo C), or Rbx1, respectively.

20. Conclusion pVHL in the E3 complex is necessary for ubiquitination of p220 and p100. Mutations found in patients abrogates this affect.

21. VHL and Hypoxia VHL tumors are usually very vascular and overproduce VEGF. They can also overproduce epo. Since VEGF and Epo are induced by hypoxia, is VHL involved in hypoxia regulation? VHL tumors are usually very vascular and overproduce VEGF. They can also overproduce epo. Since VEGF and Epo are induced by hypoxia, is VHL involved in hypoxia regulation. VHL tumors are usually very vascular and overproduce VEGF. They can also overproduce epo. Since VEGF and Epo are induced by hypoxia, is VHL involved in hypoxia regulation.

22. Cell line lacking nl VHL constitutively overproduce hypoxia-inducible mRNA. Restoration of VHL function led to suppression of hypoxia-inducible mRNAs in the presence of oxygen. Figure 1. Effect of pVHL on oxygen-regulated gene expression. mRNA analysis of RCC4 cells and stable transfectant expressing pVHL (RCC4/VHL). N, normoxia; H, hypoxia (1% O2, 16 h). VEGF, vascular endothelial growth factor; GLUT-1, glucose transporter 1; AK-3, adenylate kinase 3; TGF-beta 1, transforming growth factor-beta 1; ALD-A, aldolase A; PGK-1, phosphoglyceratekinase 1; PFK, phosphofructokinase; LDH, lactate dehydrogenase. U6 small nuclear (sn) RNA was used as an internal control. Also illustrated are two genes not influenced by VHL status or hypoxia; nuclear respiratory factor 1 (NRF-1) and beta-actin. Amount of RNA analysed is detailed in Table S1of the Supplementary Information. Cell line lacking nl VHL constitutively overproduce hypoxia-inducible mRNA. Restoration of VHL function led to suppression of hypoxia-inducible mRNAs in the presence of oxygen. Figure 1. Effect of pVHL on oxygen-regulated gene expression. mRNA analysis of RCC4 cells and stable transfectant expressing pVHL (RCC4/VHL). N, normoxia; H, hypoxia (1% O2, 16 h). VEGF, vascular endothelial growth factor; GLUT-1, glucose transporter 1; AK-3, adenylate kinase 3; TGF-beta 1, transforming growth factor-beta 1; ALD-A, aldolase A; PGK-1, phosphoglyceratekinase 1; PFK, phosphofructokinase; LDH, lactate dehydrogenase. U6 small nuclear (sn) RNA was used as an internal control. Also illustrated are two genes not influenced by VHL status or hypoxia; nuclear respiratory factor 1 (NRF-1) and beta-actin. Amount of RNA analysed is detailed in Table S1of the Supplementary Information.

23. Hypoxia-Inducible Factor Heterodimeric transcription factor consisting of HIF-1 ? and HIF-1 ?. Induces transcription of glucose transporters, glycolytic enzymes, and VEGF as well as cell-type specific proteins such as erythropoietin, inducible nitric oxide synthase, and IGF-2. HIF1- ? is required for normal vascularization, development, and survival of mouse embryos as well as for physiologic responses to chronic hypoxia in adult mice. ARE HIF and HIF related?ARE HIF and HIF related?

24. Fig. 1. VHL mediates proteasomal degradation of HIF-1 [{alpha}] under normoxic conditions. (A) Expression of HIF-1 [{alpha}] under normoxic conditions. Increasing amounts [0.2 �g (+), 0.5 �g (++), 1.0 �g (+++)] of pFLAG CMV2/HIF-1 [{alpha}] were transiently transfected into COS7 cells, and the cells were incubated for 12 h at normoxia (21% O2) or hypoxia (1% O2), as indicated. Whole-cell extracts were analyzed by immunoblotting using anti-FLAG antibodies. (B) Degradation of HIF-1 [{alpha}] in the presence of VHL. pFLAG CMV2/HIF-1 [{alpha}] was cotransfected into COS7 cells together with empty vector or wild-type VHL expression vector (pCMX/VHL). Whole-cell extracts were analyzed by immunoblotting using anti-FLAG or anti-VHL antibodies. (C) VHL mediates proteasomal degradation of HIF-1 [{alpha}] . Cells were transfected with pFLAG CMV2/HIF-1 [{alpha}] and pCMX/VHL, incubated in the absence or presence of 5 �M MG-132 for 6 h before harvesting, and cellular extracts were analyzed as in (B). (D ) VHL does not affect dioxin receptor (DR) protein levels. COS7 cells were transfected with a FLAG-tagged dioxin receptor expression vector (pCMV/DR/FLAG) in the absence or presence of pCMX/VHL, and analyzed as in (B). The mobilities of molecular weight (kDa) markers are shown on the right hand side of the blots. Fig. 1. VHL mediates proteasomal degradation of HIF-1 [{alpha}] under normoxic conditions. (A) Expression of HIF-1 [{alpha}] under normoxic conditions. Increasing amounts [0.2 �g (+), 0.5 �g (++), 1.0 �g (+++)] of pFLAG CMV2/HIF-1 [{alpha}] were transiently transfected into COS7 cells, and the cells were incubated for 12 h at normoxia (21% O2) or hypoxia (1% O2), as indicated. Whole-cell extracts were analyzed by immunoblotting using anti-FLAG antibodies. (B) Degradation of HIF-1 [{alpha}] in the presence of VHL. pFLAG CMV2/HIF-1 [{alpha}] was cotransfected into COS7 cells together with empty vector or wild-type VHL expression vector (pCMX/VHL). Whole-cell extracts were analyzed by immunoblotting using anti-FLAG or anti-VHL antibodies. (C) VHL mediates proteasomal degradation of HIF-1 [{alpha}] . Cells were transfected with pFLAG CMV2/HIF-1 [{alpha}] and pCMX/VHL, incubated in the absence or presence of 5 �M MG-132 for 6 h before harvesting, and cellular extracts were analyzed as in (B). (D ) VHL does not affect dioxin receptor (DR) protein levels. COS7 cells were transfected with a FLAG-tagged dioxin receptor expression vector (pCMV/DR/FLAG) in the absence or presence of pCMX/VHL, and analyzed as in (B). The mobilities of molecular weight (kDa) markers are shown on the right hand side of the blots.

25. Fig. 1. The purified recombinant VHL tumor suppressor complex activates HIF1 [alpha ] ubiquitination in vitro. (A) An aliquot of recombinant His-HPC4-HIF1 [alpha ] used as substrate in the ubiquitination reactions of C and D was subjected to 8% SDS/PAGE, and proteins were visualized by Coomassie staining. His-HPC4-HIF1 [alpha ] was expressed in Sf21 cells and purified by Ni2+-agarose chromatography. (B) The recombinant VHL complex was purified as described in Materials and Methods from lysates of insect cells infected with baculoviruses encoding His-VHL, Cul2, Elongin B, Elongin C, and myc-Rbx1. Aliquots of TSK DEAE-NPR column fractions were subjected to 13% SDS/PAGE, and proteins were visualized by Coomassie staining (Top). Aliquots of TSK DEAE-NPR column fractions were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure (Bottom). EloB, Elongin B; EloC, Elongin C. (C) Aliquots of TSK-DEAE-NPR column fractions indicated in the figure were assayed as described in Materials and Methods for the ability to activate HIF1 [alpha ] ubiquitination. Reactions contained [approx ] 50 ng of the His-HPC4-HIF1 [alpha ] shown in A. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1 [alpha ] conjugates (GST-Ub-HIF1 [alpha ] ) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 antibodies. (D) Aliquots of TSK-DEAE-NPR column fraction 25 were assayed as described in Materials and Methods for the ability to activate HIF1 [alpha ] or Cln2 ubiquitination activity in the presence and absence of Uba1, hUbc5a, GST-ubiquitin (GST-Ub), and ATP. Reactions contained [approx ] 50 ng of either the His-HPC4-HIF1 [alpha ] shown in A or about 50 ng of the phosphorylated Cln2 complex. Reaction products were subjected to 8% SDS/PAGE and transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 or anti-HA antibodies. (E) Aliquots of TSK-DEAE-NPR column fraction 25 were assayed as described in Materials and Methods for the ability to activate HIF1 [alpha ] ubiquitination activity in the presence of Uba1, GST-ubiquitin (GST-Ub), ATP, and approximately equimolar amounts of the E2 ubiquitin-conjugating enzymes shown in the figure ( [approx ] 100 ng hUbc5a, [approx ] 200 ng hUbc3, [approx ] 100 ng mE2-21K, [approx ] 200 ng mE2-35K, [approx ] 150 ng mE2-24K). Reactions contained [approx ] 50 ng of the His-HPC4-HIF1 [alpha ] shown in A. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1 [alpha ] conjugates (GST-Ub-HIF1 [alpha ] ) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 antibodies. Fig. 1. The purified recombinant VHL tumor suppressor complex activates HIF1 [alpha ] ubiquitination in vitro. (A) An aliquot of recombinant His-HPC4-HIF1 [alpha ] used as substrate in the ubiquitination reactions of C and D was subjected to 8% SDS/PAGE, and proteins were visualized by Coomassie staining. His-HPC4-HIF1 [alpha ] was expressed in Sf21 cells and purified by Ni2+-agarose chromatography. (B) The recombinant VHL complex was purified as described in Materials and Methods from lysates of insect cells infected with baculoviruses encoding His-VHL, Cul2, Elongin B, Elongin C, and myc-Rbx1. Aliquots of TSK DEAE-NPR column fractions were subjected to 13% SDS/PAGE, and proteins were visualized by Coomassie staining (Top). Aliquots of TSK DEAE-NPR column fractions were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure (Bottom). EloB, Elongin B; EloC, Elongin C. (C) Aliquots of TSK-DEAE-NPR column fractions indicated in the figure were assayed as described in Materials and Methods for the ability to activate HIF1 [alpha ] ubiquitination. Reactions contained [approx ] 50 ng of the His-HPC4-HIF1 [alpha ] shown in A. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1 [alpha ] conjugates (GST-Ub-HIF1 [alpha ] ) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 antibodies. (D) Aliquots of TSK-DEAE-NPR column fraction 25 were assayed as described in Materials and Methods for the ability to activate HIF1 [alpha ] or Cln2 ubiquitination activity in the presence and absence of Uba1, hUbc5a, GST-ubiquitin (GST-Ub), and ATP. Reactions contained [approx ] 50 ng of either the His-HPC4-HIF1 [alpha ] shown in A or about 50 ng of the phosphorylated Cln2 complex. Reaction products were subjected to 8% SDS/PAGE and transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 or anti-HA antibodies. (E) Aliquots of TSK-DEAE-NPR column fraction 25 were assayed as described in Materials and Methods for the ability to activate HIF1 [alpha ] ubiquitination activity in the presence of Uba1, GST-ubiquitin (GST-Ub), ATP, and approximately equimolar amounts of the E2 ubiquitin-conjugating enzymes shown in the figure ( [approx ] 100 ng hUbc5a, [approx ] 200 ng hUbc3, [approx ] 100 ng mE2-21K, [approx ] 200 ng mE2-35K, [approx ] 150 ng mE2-24K). Reactions contained [approx ] 50 ng of the His-HPC4-HIF1 [alpha ] shown in A. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1 [alpha ] conjugates (GST-Ub-HIF1 [alpha ] ) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HPC4 antibodies.

26. Fig. 3. VHL mutants that do not bind stably to Elongins B and C do not assemble into the VHL complex and do not detectably activate HIF1 [alpha ] ubiquitination. (A) Total cell lysates from Sf21 cells infected with the baculoviruses indicated in the figure were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (B) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (C) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were assayed for the ability to activate HIF1 [alpha ] ubiquitination as described in Materials and Methods. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1 [alpha ] conjugates (GST-Ub-HIF1 [alpha ] ) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HIF1 [alpha ] antibodies. EloB, Elongin C; EloC, Elongin C. Fig. 3. VHL mutants that do not bind stably to Elongins B and C do not assemble into the VHL complex and do not detectably activate HIF1 [alpha ] ubiquitination. (A) Total cell lysates from Sf21 cells infected with the baculoviruses indicated in the figure were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (B) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (C) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were assayed for the ability to activate HIF1 [alpha ] ubiquitination as described in Materials and Methods. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1 [alpha ] conjugates (GST-Ub-HIF1 [alpha ] ) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HIF1 [alpha ] antibodies. EloB, Elongin C; EloC, Elongin C.

27. Hypoxia-Inducible Factor cont. Under normoxic conditions HIF-1? is post-translationally hydroxylated at proline residues.

30. Our Patient Atypical in that she has an ACTH expressing tumor. Pathologically this resembles a paraganglioma. She is followed by CT scan to assess growth of liver nodules and development of renal cell carcinoma. She has had stable to decreased disease over the last year and a half.