| 1. |
- Lindström, Lisa, et al.
(författare)
-
Therapeutic Targeting of Nuclear Gamma-Tubulin in RB1-negative Tumors.
- 2015
-
Ingår i: Molecular Cancer Research. - 1557-3125. ; 13:7, s. 1073-1082
-
Tidskriftsartikel (refereegranskat)abstract
- In addition to its cytosolic function, gamma-tubulin is a chromatin-associated protein. Reduced levels of nuclear gamma-tubulin increase the activity of E2 promoter-binding factors (E2F) and raise the levels of retinoblastoma (RB1) tumor suppressor protein. In tumor cells lacking RB1 expression, decreased gamma-tubulin levels induce cell death. Consequently, impairment of the nuclear activity of gamma-tubulin has been suggested as a strategy for targeted chemotherapy of RB1-deficient tumors; thus, tubulin inhibitors were tested to identify compounds that interfere with gamma-tubulin. Interestingly, citral increased E2F activity but impaired microtubule dynamics while citral analogs, like citral dimethyl acetal (CDA), increased E2F activity without affecting microtubules. The cytotoxic effect of CDA on tumor cells was attenuated by increased expression of either RB1 or gamma-tubulin, and increased by reduced levels of either RB1 or gamma-tubulin. Mechanistic study, in silico and in vitro, demonstrated that CDA prevents GTP binding to gamma-tubulin and suggested that the FDA approved drug dimethyl fumarate is also a gamma-tubulin inhibitor. Finally, in vivo growth of xenograft tumors carrying defects in the RB1 signaling pathway were inhibited by CDA treatment. These results demonstrate that inhibition of gamma-tubulin has the potential to specifically target tumor cells and may aid in the design of safer and more efficient chemotherapeutic regimes.
|
|
| 2. |
- Mohlin, Frida, et al.
(författare)
-
Functional characterization of two novel non-synonymous alterations in CD46 and a Q950H change in factor H found in atypical hemolytic uremic syndrome patients.
- 2015
-
Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 65:2, s. 367-376
-
Tidskriftsartikel (refereegranskat)abstract
- Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation, characterized by hemolytic anemia, thrombocytopenia and acute renal failure. Mutations in complement inhibitors are major risk factors for development of aHUS. The three aHUS patients reported in this study had several previously identified alterations in complement inhibitors; e.g. risk haplotypes in CD46 and factor H but we also identified two novel heterozygous non-synonymous CD46 alterations (p.E142Q and p.G259V). Presence of G259V caused decreased expression of the recombinant mutant CD46 compared to wild type (WT). Western blot analysis showed that the majority of the expressed G259V protein was in the precursor form, suggesting that it is processed less efficiently than WT. Low CD46 expression on the surface of the patient's neutrophils confirmed the in vitro results. Further, G259V had a substantially impaired ability to act as a cofactor to factor I, in the degradation of both C3b and C4b. The E142Q mutant showed neither decreased expression nor impaired function. Two of the patients also had a heterozygous non-synonymous alteration in factor H (p.Q950H), reported previously in aHUS but not functionally tested. This variant showed moderately impaired function in hemolytic assays, both using patient sera and recombinant proteins. The recombinant Q950H also showed a somewhat decreased expression compared to WT but the complement inhibitory function in fluid phase was normal. Taken together, we report a novel CD46 alteration showing both a decreased protein expression and substantially impaired cofactor function (G259V) and another without an effect on expression or cofactor function (E142Q). Moreover, mild consequences of a previously reported aHUS associated rare variant in factor H (Q950H) was also revealed, underlining the clear need for functional characterization of each new aHUS associated mutation.
|
|
| 3. |
- Somajo, Sofia, et al.
(författare)
-
Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction
- 2015
-
Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 113:05, s. 976-987
-
Tidskriftsartikel (refereegranskat)abstract
- Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50–60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LGdomains.
|
|
