Lipofuscin inhibits the proteasome by binding to surface motifs

Lipofuscin, a highly oxidized aggregate, consists of covalently cross-linked proteins, lipids, and sugar residues and is one of the major life-span-limiting factors in postmitotic aging cells. An artificial model of this material, showing characteristics and effects comparable to those of the natural form, has turned out to be very useful for in vitro studies. Artificial lipofuscin was used to investigate its effects on the viability of human fibroblasts, its rate of uptake, and its ability to inhibit the proteasomal system. The inhibition of the proteasomal system is one of the major aspects of the cytotoxic effects of lipofuscin. We present here that this proteasomal inhibition is due to proteasomal binding to the lipofuscin surface motifs, degradable by protease K. Furthermore, removal of the surface peptide structures by protease K strongly reduces the cytotoxic effects of lipofuscin and binding of cellular proteins and proteasomes to intracellular protein aggregates.     

Protein conformational changes of Agrobacterium phytochrome Agp1 during chromophore assembly and photoconversion

Phytochromes are widely distributed photochromic biliprotein photoreceptors. Typical bacterial phytochromes such as Agrobacterium Agp1 have a C-terminal histidine kinase module; the N-terminal chromophore module induces conformational changes in the protein that lead to modulation of kinase activity. We show by protein cross-linking that the C-terminal histidine kinase module of Agp1 mediates stable dimerization. The fragment Agp1-M15, which comprises the chromophore module but lacks the histidine kinase module, can also form dimers. In this fragment, dimer formation was stronger for the far-red-absorbing form Pfr than for the red-absorbing form Pr. The same or similar behavior was found for Agp1-M15Delta9N and Agp1-M15Delta18N, which lack 9 and 18 amino acids of the N-terminus, respectively. The fragment Agp1-M20, which is derived from Agp1-M15 by truncation of the C-terminal "PHY domain" (191 amino acids), can also form dimers, but dimerization is independent of irradiation conditions. The cross-linking data also showed that the PHY domain is in tight contact with Lys 16 of the protein and that the nine N-terminal amino acids mediate oligomer formation. Limited proteolysis shows that the hinge region between the chromophore module and the histidine kinase and a part of the PHY domain become exposed upon Pr to Pfr photoconversion.     

Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome

Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory d-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.     

Activation and polar sequestration of PopA,a c‐di‐GMP effector protein involved in Caulobacter crescentus cell cycle control

When Caulobacter crescentus enters S‐phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co‐option as c‐di‐GMP effector protein. While the C‐terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N‐terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N‐terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c‐di‐GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S‐phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.     

EGFR-Targeted TRAIL and a Smac Mimetic Synergize to Overcome Apoptosis Resistance in KRAS Mutant Colorectal Cancer Cells

TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed Db_αEGFR-scTRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of Db_αEGFR-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward Db_αEGFR-scTRAIL in these 3D cultures. We show that the antibody moiety of Db_αEGFR-scTRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing Db_αEGFR-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects Db_αEGFR-scTRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-Ras^G12V. In the presence of doxycycline, these cells showed increased resistance to Db_αEGFR-scTRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and Flip_S. Co-treatment of cells with the Smac mimetic SM83 restored the Db_αEGFR-scTRAIL-induced apoptotic response. Importantly, this synergy between Db_αEGFR-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that Db_αEGFR-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.     

Detection of IGF2BP3, HOXB7, and NEK2 mRNA Expression in Brush Cytology Specimens as a New Diagnostic Tool in Patients with Biliary Strictures

Introduction

It is a challenging task to distinguish between benign and malignant lesions in patients with biliary strictures. Here we analyze whether determination of target gene mRNA levels in intraductal brush cytology specimens may be used to improve the diagnosis of bile duct carcinoma.

Materials and Methods

Brush cytology specimens from 119 patients with biliary strictures (malignant: n = 72; benign: n = 47) were analyzed in a retrospective cohort study. mRNA of IGF-II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), Forkhead box M1 (FOXM1), kinesin family member 2C (KIF2C) and serine/threonine kinase NEK2 was determined by semi-quantitative RT-PCR using the ΔCt method.

Results

IGF2BP3 (p<0.0001), HOXB7 (p<0.0001), and NEK2 (p<0.0001) mRNA expression levels were significantly increased in patients with cholangiocarcinoma or pancreatic cancer. Median ΔCt values differed by 3.5 cycles (IGF2BP3), 2.8 cycles (HOXB7) and 1.3 cycles (NEK2) corresponding to 11-fold, 7-fold and 2.5-fold increased mRNA levels in malignant versus benign samples. Sensitivity to detect biliary cancer was 76.4% for IGF2BP3 (80.9% specificity); 72.2% for HOXB7 (78.7% specificity) and 65.3% for NEK2 (72.3% specificity), whereas routine cytology reached only 43.1% sensitivity (85.4% specificity). Diagnostic precision was further improved, when all three molecular markers were assessed in combination (77.8% sensitivity, 87.2% specificity) and achieved 87.5% sensitivity and 87.2% specificity when molecular markers were combined with routine cytology.

Conclusions

Our data suggest that measuring IGF2BP3, HOXB7 and NEK2 mRNA levels by RT-PCR in addition to cytology has the potential to improve detection of malignant biliary disorders from brush cytology specimens.     

La autoantigen suppresses IRES-dependent translation of the hepatitis A virus

The human RNA-binding protein La, is an essential trans-acting factor in IRES-dependent translation initiation of poliovirus, the prototypic picornavirus. For hepatitis A virus (HAV), an unusual member of this virus family, the role of host proteins in its inefficient translation and slow replication is unclear. Using small interfering RNA in vivo and purified La in vitro, we demonstrate for the first time that La suppresses HAV IRES-mediated translation and replication. We show that La binds specifically to distinct parts of the HAV IRES and that—unlike poliovirus—HAV proteinase 3C does not cleave La. The La-mediated suppression of HAV translation and stimulation of poliovirus translation implies unexpected mechanistic differences between viral IRES elements.     

17beta-Estradiol inhibits osteoprotegerin production by the estrogen receptor-alpha-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p < 0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.     

Subpicosecond midinfrared spectroscopy of the Pfr reaction of phytochrome Agp1 from Agrobacterium tumefaciens

Phytochromes are light-sensing pigments found in plants and bacteria. For the first time, the P_fr photoreaction of a phytochrome has been subject to ultrafast infrared vibrational spectroscopy. Three time constants of 0.3 ps, 1.3 ps, and 4.0 ps were derived from the kinetics of structurally specific marker bands of the biliverdin chromophore of Agp1-BV from Agrobacterium tumefaciens after excitation at 765 nm. VIS-pump-VIS-probe experiments yield time constants of 0.44 ps and 3.3 ps for the underlying electronic-state dynamics. A reaction scheme is proposed including two kinetic steps on the S₁ excited-state surface and the cooling of a vibrationally hot P_fr ground state. It is concluded that the upper limit of the E-Z isomerization of the C₁₅ = C₁₆ methine bridge is given by the intermediate time constant of 1.3 ps. The reaction scheme is reminiscent of that of the corresponding P_r reaction of Agp1-BV as published earlier.