Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome <i>c</i>

Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome <i>c</i> (cyt <i>c</i>), ligation of Met80 to the heme iron is critical for the protein’s electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met is, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in the maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, nuclear magnetic resonance, and electron paramagnetic resonance spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt <i>c</i>. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation, and the pH-dependent changes in ligation provide a detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling ligation of the native Met80 to the heme iron

Intermittent Induction of HIF-1α Produces Lasting Effects on Malignant Progression Independent of Its Continued Expression

<div><p>Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of <i>HIF1A</i> in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor β-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense.</p></div

Intermittent induction of HIF1α(PP) promoted intracranial spread of U-87 MG cells.

<p>Bioluminescent imaging analysis of β-gal and HIF1α(PP)-derived intracranial tumors (A) and HIF2α(PP)-derived intracranial tumors (C). The respective tumor volumes were calculated based on the relative luminescent units (RLU) and plotted in a log scale (B and D). *, <i>p</i>-value < 0.05. (E) HIF1α(PP)-derived tumors had small yet numerous lesions invading the Ammon’s horn of the hippocampal region (XG026) and hindbrain and cerebellum (XG042), as indicated by arrowheads. (F) β-gal- and HIF2α(PP)-derived tumors were large and often singular in the cerebral cortex. Tumor lesions are demarcated in dash lines. Hematoxylin and eosin—stained images are presented at 25× and 200× magnifications, with scale bars of 1 mm and 100 μm, respectively.</p

Tetracycline regulation of HIF1α(PP) and HIF2α(PP) expression and transcriptional activity.

<p>(A) Tetracycline regulation is diagrammed where the addition of tetracycline (tet) results in dissociation of tetracycline repressor (TetR) from the tetracycline operon (TetO) and, in turn, gene activation. (B) Western blot analysis of transduced cell types, as indicated, for the expression of HIF1α(PP) and HIF2α(PP) after 2-day treatment with tetracycline. (C) Transcriptional activities of HIF1α(PP) and HIF2α(PP) were tested in a reporter assay in reference to β-galactosidase (β-gal). ***, <i>p</i>-value < 0.001. (D, E) The expression of HIF target genes (<i>PGK1</i>, <i>CA9</i>, <i>VEGFA</i>, and <i>LOX</i>) was analyzed in specified cell lines by using real-time PCR after 2-day treatment with tetracycline.</p

Intermittent induction resulted in loss of of HIF1α(PP) expression.

<p>(A) Intermittent induction involves the administration of tetracycline into cell culture each week on day 1 and removal on day 4 each week for a total of 8 weeks. Afterwards, cells were allowed to expand for further analyses and injections. (B) After intermittent induction (8W), different types of cells as indicated were induced again with tetracycline for 2 days and analyzed by Western blotting in reference to those without intermittent induction (0W). (C) Cell proliferation was determined by cell counting after intermittent induction. ***, <i>p</i>-value < 0.001.</p

U-2 OS cells acquired tumorigenicity after intermittent induction of HIF1α(PP).

<p>(A) Tumor incidence is shown in 6 NSG mice after bilateral, subcutaneous injections of the 8-week HIF1α(PP) and HIF2α(PP) cells. (B) Only injections of the HIF1α(PP) cells produced tumors, as indicated by arrowheads. Scale bar, 1 cm. (C) Tumor volume was calculated based on measurements and plotted as a function of time. ***, <i>p</i>-value < 0.001. (D) Hematoxylin and eosin staining of the tumor specimens reveals invasion of the dermal layer (<i>a</i>), numerous mitoses (arrowheads) (<i>b</i>), necrosis (N) (<i>c</i>), and invasion into the striated muscle layer (<i>d</i>). Scale bar, 100 μm.</p

Rictor deficiency altered the cell cycle of the mature B cells in BM.

<p>(<b>A</b>) Flow cytometry analysis of committed B cell apoptosis (n = 5). (B) The representative cell cycle of committed B cells from control and <i>Rictor</i>-deleted mice (n = 5). (C), (D), (E), and (F) Cell cycle analysis of pro-B, pre-B, immature B, and mature B cells (n = 5). All of these experiments were performed in triplicate (*: P<0.05; **: P<0.01; ***: P<0.001).</p

Rictor deletion impaired early B cell development in BM.

<p>(A) Representative flow cytometry profile of hematopoietic stem cells and progenitor cells based on the indicated surface marker. (B) Total BM cell count. (C) and (D) The percentages of stem and progenitor cells were analyzed by flow cytometry after 6 months of pIpC treatment (n = 5). (E) The representative flow cytometry profile for committed B cells in BM. (F) The percentage of committed B cells in BM after 6 months of pIpC treatment (n = 5). All of these experiments were performed in triplicate (*: P<0.05, **: P<0.01, ***: P<0.001).</p

Rictor deletion leads to a decrease in B cells in the PB and spleen.

<p>(A) 1×10⁵ LT-HSC, ST-HSC, MPP, CLP, CMP, GMP, MEP, T, B and myeloid cells were sorted with FACS for real-time PCR. The data shown have been normalized to GAPDH. (B) <i>Rictor</i> excision in BM cells. BMMNCs were sorted fromMx-1 Cre⁺ Rictor^fl/fl micethat did or did not undergo pIpC treatment, and PCR was performed using two different sets of primers to detect <i>Rictor</i> in WT (<i>Rictor</i>⁺), conditional (<i>Rictor</i>^fl) and deleted (<i>Rictor</i>^Δ) alleles. (C) Rictor is efficiently deleted in the BM of Mx-1 Cre⁺ Rictor^fl/fl mice with pIpC treatment for 1 month or 6 months. One or 6 months after pIpC treatment, 1×10⁶BMMNCs were isolated from Mx-1 Cre⁺Rictor^fl/flmice for real-time PCR. (D) Rictor was efficiently deleted in HSCs and HPCs. One month after pIpC treatment, 1×10⁵HSC, HPC and Lin⁻ cells were sorted with FACS for real-time PCR. The data shown were normalized to GAPDH. (E) and (G) The PB counts of control and Rictor-deleted mice1 and 6 months after pIpC treatment (n = 10). (F) and (H) The percentage of T (CD3⁺), B (B220⁺) and myeloid cells (Mac-1⁺) in PB were analyzed after 1 and 6 months of pIpC treatment by flow cytometry (n = 10). (I) The total number of spleen cells in the control and Rictor-deleted mice were counted using a hemocytometer (n = 5). (J) The percentage of B cells in the spleen was analyzed by flow cytometry (n = 5). The flow cytometry and hemocytometry measurements were performed in triplicate(*: P<0.05; **: P<0.01; ***: P<0.001).</p

Knockdown of FoxO1 in Rictor-deficient HSPCs increased the number of B cells in PB and BM.

<p>(A) Diagram of the experimental design. <i>Rictor</i>-deficient HSPCs transduced by FoxO1 and control shRNA (GFP⁺) were transplanted into recipient mice. (B) The percentages of T, B, and myeloid cells derived from the control and FoxO1 knockdown cells (GFP⁺) in PB were analyzed by flow cytometry (n = 5). (C) The percentage of committed B cells derived from control and FoxO1 knockdown cells (GFP⁺) was detected by flow cytometry (n = 5). (D) The GFP⁺ B cells from the BM of recipients were sorted and lysed for RNA extraction. The expression of FoxO1, IL-7R, and Rag-1 at the mRNA level was analyzed by real-time PCR. (E) The GFP⁺ B cells from the BM of recipients were sorted and lysed for Western blotting. The expression levels of Rictor in the FoxO1 knockdown GFP⁺ B cells were normalized to Actin and are presented as the fold increase relative to that of the control GFP⁺ B cells. All of these experiments were performed in duplicate (*: P<0.05, **: P<0.01, ***: P<0.001).</p