Figure 2: New TCEB1 mutations and HIF accumulation. | Nature Genetics

Figure 2: New TCEB1 mutations and HIF accumulation.

From: Integrated molecular analysis of clear-cell renal cell carcinoma

Figure 2

(a) Two examples of cullin–RING ubiquitin ligase system molecular assemblies using CUL2 or CUL5 (left) and CUL3 (right) that interact with the BC-box protein–Elongin C–Elongin B complex and BTB protein, respectively, to recruit substrate for ubiquitination and subsequent degradation. VHL and KEAP1 are examples of BC-box and BTB proteins, respectively, that recruit HIF and NRF2 proteins for ubiquitin-mediated degradation. (b) TCEB1 mutations (8 of 240 tumors) affect the domains for binding to VHL in Elongin C. (ce) Structure of the VHL complex comprising Elongin B, Elongin C and VHL (c), with the positions of mutated amino acids Tyr79 (d) and Ala100 (e) in Elongin C indicated. (f,g) A critical hydrogen bond between Tyr79 in Elongin C and Pro154 in VHL (d) was predicted to be abolished in the Tyr79Cys (f) and Tyr79Ser (g) Elongin C mutants. (h) Hydrophobic binding around Ala100 in Elongin C and Val165 and Val166 in VHL (g) could be compromised in the Ala100Pro Elongin C mutant. (i) Protein blotting for the indicated components of the VHL-CUL2 complex in total cell lysates (left) and in lysates after immunoprecipitation (IP) with antibody to HA (Elongin C) (right). Lysates were from HEK 293T cells transduced with mock, wild-type (WT) or mutant TCEB1 constructs (encoding Tyr79Cys and Ala100Pro Elongin C). (j) Protein blotting for the effect of TCEB1 (Elongin C) mutations on HIF accumulation using non-specific siRNA (left) or siRNA specific for endogenous TCEB1 (right). Endogenous and exogenous Elongin C and HIF-1α were examined. Exogenous 3′ HA–tagged Elongin C was detected with an antibody to HA. Note that antibody to Elongin C could discriminate slower migrating exogenous protein (single arrowhead) from faster migrating endogenous protein (double arrowheads). (k) Immunohistochemical analysis of HIF-1α and HIF-2α expression in representative cases from primary ccRCC specimens with TCEB1 mutations (n = 5), VHL mutations (n = 92) or without TCEB1 or VHL mutations (n = 9) as well as in normal kidney tissue (n = 1). Red and black arrows indicate positive and negative nuclear immunoreactivity, respectively. Scale bars, 20 μm.

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