Susan R. Schoen

Ubiquitination of a Novel Deubiquitinating Enzyme Requires Direct Binding to von Hippel-Lindau Tumor Suppressor Protein

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of theVHL gene. Recent studies suggest that VHL protein (pVHL) is a component of an E3 ubiquitin ligase, but the detailed biological function of pVHL remains to be determined. To further elucidate the biological functions of pVHL, we searched pVHL-interacting proteins using yeast two-hybrid screening. A novel protein named VHL-interacting deubiquitinating enzyme 1 (VDU1) was identified as being able to directly interact with pVHL in vitro and in vivo. We have determined the full-length cDNA of this enzyme, which includes two putative subtypes. Type I consists of 942 amino acids, and type II consists of 911 amino acids with predicted molecular masses of 107 and 103 kDa, respectively. We have also cloned a mouse homologue of this enzyme. Sequence analysis reveals that this protein is conserved between human and mouse and contains the signature motifs of the ubiquitin-specific processing protease family. Enzymatic function studies demonstrate its deubiquitinating activity. We have determined that the VDU1-interacting region in pVHL is located in its β-domain, and several naturally occurring mutations located in this domain disrupt the interaction between pVHL and VDU1 protein. Co-immunoprecipitation demonstrates that VDU1 can be recruited into the pVHL-elongin C-elongin B complex. Finally, we demonstrate that VDU1 is able to be ubiquitinated via a pVHL-dependent pathway for proteasomal degradation, and VHL mutations that disrupt the interaction between VDU1 and pVHL abrogate the ubiquitination of VDU1. Our findings indicate that VDU1, a novel ubiquitin-specific processing protease, is a downstream target for ubiquitination and degradation by pVHL E3 ligase. Targeted degradation of VDU1 by pVHL could be crucial for regulating the ubiquitin-proteasome degradation pathway.

Identification of a deubiquitinating enzyme subfamily as substrates of the von Hippel-Lindau tumor suppressor

The VHL protein (pVHL) is a component of an E3 ubiquitin ligase complex which is involved in the ubiquitination and degradation of the alpha subunits of HIF (hypoxia-inducible factor) in the presence of oxygen. However, it is of considerable interest to identify pVHL substrates other than HIF. In our previous studies, we have shown that VDU1 (pVHL-interacting deubiquitinating enzyme-1) can be ubiquitinated for rapid degradation in a pVHL-dependent manner. In this report we show that another uncharacterized deubiquitinating enzyme, named VDU2 (pVHL-interacting deubiquitinating enzyme-2), is a substrate of pVHL. Based on human and mouse cDNA sequences, VDU1 and VDU2 are identical in approximately 59% of the amino acids with strong homology in the N-terminus and C-terminus and a weaker similarity in the middle region. VDU2 contains the signature motifs of the ubiquitin-specific processing protease family and possesses deubiquitinating activity. Like VDU1, VDU2 interacts with pVHL beta-domain and these two proteins can compete with each other to bind to pVHL. Finally, we demonstrate that VDU2 can also be ubiquitinated and degraded in a pVHL-dependent manner. Based on their amino acid sequence homology and functional interaction with pVHL, VDU1 and VDU2 define a subfamily of ubiquitin specific processing proteases. Since deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination-related processes, VDU1 and VDU2 could be important substrates of pVHL E3 ligase complex.

The VHL protein recruits a novel KRAB‐A domain protein to repress HIF‐1α transcriptional activity

The von Hippel‐Lindau tumor suppressor (pVHL) is a component of an E3 ubiquitin ligase and targets hypoxia‐inducible factor‐1α (HIF‐1α) for ubiquitylation and degradation under normoxic conditions. pVHL also directly inhibits HIF‐1α transactivation by recruiting histone deacetylases. Here, we report a novel pVHL‐interacting protein that functions as a negative regulator of HIF‐1α transactivation. This protein, generated from the ZnF197 locus by alternative splicing, contains a Kruppel‐associated box (KRAB)‐A domain and a SCAN domain, but lacks the 22 C2H2‐type zinc fingers present in ZnF197. Therefore, we named this protein pVHL‐associated KRAB‐A domain‐containing protein (VHLaK). We demonstrate that the KRAB‐A domain in VHLaK mediates pVHL binding and functions as a transcriptional repression module. The SCAN domain mediates VHLaK homo‐oligomerization, which enhances VHLaK repressive activity. pVHL can recruit VHLaK to repress HIF‐1α transcriptional activity and HIF‐1α‐induced VEGF expression. Finally, we demonstrate that pVHL, VHLaK and KAP1/TIF‐1β can be recruited into a single complex, indicating that KAP1/TIF‐1β may participate in pVHL‐mediated transcriptional repression of HIF‐1α. Our findings provide a novel mechanism for the modulation of HIF‐1α transactivation by pVHL.

Identification of the RNA polymerase II subunit hsRPB7 as a novel target of the von Hippel—Lindau protein

Inactivation of the von Hippel—Lindau (VHL) tumor suppressor gene is linked to the hereditary VHL disease and sporadic clear cell renal cell carcinomas (CCRCC). VHL‐associated tumors are highly vascularized, a characteristic associated with overproduction of vascular endothelial growth factor (VEGF). The VHL protein (pVHL) is a component of the ubiquitin ligase E3 complex, targeting substrate proteins for ubiquitylation and subsequent proteasomic degradation. Here, we report that the pVHL can directly bind to the human RNA polymerase II seventh subunit (hsRPB7) through its β‐domain, and naturally occurring β‐domain mutations can decrease the binding of pVHL to hsRPB7. Introducing wild‐type pVHL into human kidney tumor cell lines carrying endogenous mutant non‐functional pVHL facilitates ubiquityl ation and proteasomal degradation of hsRPB7, and decreases its nuclear accumulation. pVHL can also suppress hsRPB7‐induced VEGF promoter transactivation, mRNA expression and VEGF protein secretion. Together, our results suggest that hsRPB7 is a downstream target of the VHL ubiquitylating complex and pVHL may regulate angiogenesis by targeting hsRPB7 for degradation via the ubiquitylation pathway and preventing VEGF expression.

Calcitonin receptor mRNA expression in the human prostate

OBJECTIVES A subpopulation of prostate neuroendocrine (NE) cells contain calcitonin (CT). It has been postulated that CT-producing cells in the prostate account for the high CT level in the semen, and may be involved in the regulation of other epithelial cells via a paracrine mechanism. The presence of CT binding sites in the plasma membrane fraction of prostate tissue has been demonstrated by radioligand binding assay. In the present study, we investigated the CT receptor gene expression in the human prostate, a key component of the autocrine/paracrine loop in the CT functional pathway. METHODS Reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate the CT receptor mRNA expression in normal prostate tissue. Subsequent DNA sequencing was used to verify RT-PCR amplified products and to determine the isoform of the receptor. To define the location of the CT receptor expression, nonradioactive in situ hybridization was performed with a digoxigenin-labeled probe complementary to the coding region of the CT receptor mRNA. A polyclonal antibody against CT was used to reveal the CT-secreting cells in the prostate. RESULTS CT receptor MRNA expression was detected in the prostate tissue. Further analysis of the DNA sequence showed that CT receptor expressed in the prostate was the isoform without a 16-amino acid insert in the first intracellular domain. In situ hybridization revealed that CT receptor was present in the prostate NE cells. Immunocytochemical staining of mirror image sections showed that some CT-secreting cells also expressed CT receptor. CONCLUSIONS CT receptor expression in the prostate, a key component in the CT functional pathway, is located in subsets of dispersed NE cells (CT secreting and CT nonsecreting), which indicates that prostate CT may play an important role in the autocrine/paracrine regulation of the prostate NE system.

Suramin inhibits gonadotropin action in rat testis: implications for treatment of advanced prostate cancer.

Suramin is being evaluated for the treatment of metastatic prostate cancer based on its inhibition of growth factor action. In addition, suramin may inhibit the endocrine control of androgen production, which was explored herein. Adult Sprague-Dawley rats were injected (i.p.) daily with varying doses of suramin. At a cumulative dose of 200 mg., suramin significantly depressed serum testosterone (p less than 0.05), and follicle stimulating hormone (p less than 0.002) levels. In vitro studies showed that suramin-mediated suppression of androgen production might be secondary to inhibition of gonadotropin action. In MA-10 cell cultures, suramin inhibited a maximum stimulatory dose of human chorionic gonadotropin with an ED50 of 4.4 microM. Studies in rat Sertoli cell cultures showed that follicle stimulating hormone action was also inhibited by suramin, with an ED50 of 8.6 microM. Using receptor binding assays with calf testis membrane, we showed that suramin inhibited 125I-hFSH binding to receptor in a dose dependent fashion with an ED50 of 10.4 microM; comparable to the ED50 of suramin inhibition of follicle stimulating hormone action in Sertoli cell culture cells. Thus the mechanism of suramins suppression of androgen production may involve multiple sites of action, including inhibition of gonadotropin binding to its receptor and suppression of pituitary gonadotropin levels in serum. This inhibition of androgen production may be useful in the treatment of advanced prostate cancer.

Clinical association with urinary glycosaminoglycans and urolithiasis

OBJECTIVES To determine the clinical association between urinary glycosaminoglycan (GAG) concentration and kidney stone disease. METHODS Thirty-five patients (14 women and 21 men) with a history of stone disease and 37 controls (13 women and 24 men) were evaluated for urinary GAG concentration. By using a new dye-binding assay, the total GAG concentration in the urine was measured and corrected to urinary creatinine levels (micrograms of GAG per milligram creatinine). RESULTS The mean urinary GAG concentration in those with stones was significantly lower (31.5 +/- 2.6 microg GAG/mg creatinine) than in the controls (43.8 +/- 3.8 microg GAG/mg creatinine, P = 0.01). Male patients with stones also had a significantly lower mean GAG concentration (26.1 +/- 1.8) than did the female patients (39.6 +/- 5.3, P = 0.009). The mean GAG concentration between ureteral (n = 13) versus renal (n = 22), single (n = 19) versus multiple (n = 16), family history (n = 11) versus no family history (n = 24), large (n = 13) versus small (n = 20), and the presence (n = 22) versus absence (n = 13) of residual stones did not show any significant differences. However, patients with recurrent stone formation (n = 21) had significantly lower mean GAG levels (26.4 +/- 1.6) compared with those with single stone formation (n = 14; 39.2 +/- 5.5, P = 0.01). CONCLUSIONS Lower urinary GAG levels are more common in patients with stone formation. This may play a more determinant role in male patients and those with recurrent stone formation.

Effect of Intravesical Administration of Tumor Necrosis Serum and Human Recombinant Tumor Necrosis Factor on a Murine Bladder Tumor

The effect of intravesical administration of tumor necrosis factor (TNF) on the implantability and growth of bladder cancer was studied using the murine tumor model, MBT-2. Crude TNF was prepared from serum (TNS) of BCG infected rats after injection of endotoxin or the recombinant product of human TNF-alpha was used. Treatment was begun 24 hr. or seven days after tumor instillation and repeated three times. Tumor implantability and tumor weight were determined on day 21. Low dose TNF (200 U), given in the form of TNS, failed to suppress the growth of established tumors (seven-day tumor). It did, however, significantly reduce tumor implantability. The growth of seven-day tumors was significantly suppressed by administration of higher dose of TNF (3,700 U) given in recombinant form.

Disruption of tubular Flcn expression as a mouse model for renal tumor induction

The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression of tumorigenesis. The mTOR and TGF-β signalings were upregulated in Flcn-deficient tumors, and these two activated pathways may synergetically cause renal tumorigenesis. Treatment of knockout mice with the mTOR inhibitor rapamycin for 10 months led to the suppression of tumor growth. Thus, our model recapitulates human Birt-Hogg-Dubé kidney tumorigenesis, provides a valuable tool for further study of Flcn-deficient renal tumorigenesis, and tests new drugs/approaches to their treatment.

Effects of sympathetic denervation of rat internal genitalia on daily sperm output

Using the technique of vasocystostomy, daily sperm output was determined in rats undergoing surgical removal of the sympathetic nerves to the internal genitalia. Between ten and fourteen days after denervation, the daily sperm output significantly (p less than 0.001) decreased below presurgical rates and those of sham-operated control animals (p less than 0.01). This change occurred without changes in plasma testosterone concentration or testicular histology. These data suggest neural control of sperm transport in the rat.