FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX

Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/β, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/β modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance.     

A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation

The ESX-1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis and M. marinum. To date, three proteins are known to be secreted by ESX-1 and necessary for virulence, two of which are CFP-10 and ESAT-6. The ESX-1 secretion and the virulence mechanisms are not well understood. In this study, we have examined the M. marinum secretomes and identified four proteins specific to ESX-1. Two of those are CFP-10 and ESAT-6, and the other two are novel: MM1553 (homologous to Rv3483c) and Mh3881c (homologous to Rv3881c). We have shown that Mh3881c, CFP-10 and ESAT-6 are co-dependent for secretion. Mh3881c is being cleaved at close to the C-terminus during secretion, and the C-terminal portion is critical to the co-dependent secretion, the ESAT-6 cellular levels, and interaction with ESAT-6. The co-dependent secretion is required for M. marinum intracellular growth in macrophages, where the Mh3881c C-terminal portion plays a critical role. The role of the co-dependent secretion in intracellular growth correlates with its role in inhibiting phagosome maturation. Both the secretion and the virulence defects of the Mh3881c mutant are complemented by Mh3881c or its M. tuberculosis homologue Rv3881c, suggesting that in M. tuberculosis, Rv3881c has similar functions.     

Rapid and sensitive detection of an intracellular pathogen in human peripheral leukocytes with hybridizing magnetic relaxation nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample's magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen.     

Molecular identification and properties of a light-insensitive rice catalase-B expressed in E. coli

Catalase plays a central role in plant stress responses but is highly susceptible to photoinhibition. A rice catalase-B protein avoiding photoinhibition was developed by mutagenesis of specific amino acids: Leu-189 to Trp-189 and His-225 to Thr-225 and then recombinantly expressed in E. coli. In addition, the site specific mutation also induced 2–2.5-fold increase in enzyme velocity with high affinity for its substrate and showed nearly a 3-fold lower K _m than the wild protein. These characteristic of mutated rice catalase-B is highly promising in transgenic research to increase plant productivity under stress conditions.     

Cytotoxic effector T cells elicited by the killed tumor vaccine differ significantly from the effectors generated during active growth of a murine B-cell lymphoma

Active specific immunotherapy of neoplastic diseases is an elusive goal. Using a murine B lymphoma 2C3, we showed that vaccination with the killed tumor cells effectively induces protective immunity and a cytotoxic T cell (CTL) response. Similar protection, however, is rarely observed in mice bearing live tumor cells. These animals usually succumb to the progressively growing tumor. In this study, we inquired whether the splenic CTL induced during tumor progression in mice differ from those evoked by the killed tumor cells. Here we demonstrate that the CTL generated following vaccination are significantly different from those induced in the tumor-bearing hosts. Adding to the complexity, the CTL from the early tumor bearers also differ significantly from those induced at the late stages. These differences are based on their cytotoxic activity, MHC allele specificity, mitogen responsiveness, cytokine secretion profile and T cell receptor Vβ gene expression. The results clearly indicate that passive immunization with killed tumor is most effective, possibly because the CTL induced are not subject to the same regulatory pressure as those induced during active tumor growth. This decreasing effectiveness of CTL could be due to greater variability in antigenic stimulus, less involvement of innate immunity, changes in cytokine milieu and/or costimulatory factors. Received: 5 February 1999 / Accepted: 22 June 1999     

Detection of an intermediate during unfolding of bacterial cell division protein FtsZ: loss of functional properties precedes the global unfolding of FtsZ

Using environment-sensitive fluorescence of 1-anilinonaphthalene-8-sulfonic acid, polarization of fluorescein 5'-isothiocyanate-labeled FtsZ, and far-UV circular dichroism spectroscopy, the chemical unfolding of FtsZ was found to proceed through two steps. The first step of the urea-induced unfolding produced an intermediate, which then unfolded at higher concentrations of urea. The intermediate state contains native-like secondary structure and much less tertiary structure compared with the native state. It is distinct from the native state as well as from the unfolded state. Similar to urea-induced unfolding of FtsZ, thermal unfolding of FtsZ also occurs in two steps. The midpoints for the first and second thermal unfolding transitions were found to be 38 +/- 4 and 77 +/- 5 degrees C, respectively. Further, the functional properties of FtsZ are extremely sensitive to urea, guanidium chloride, and sodium dodecyl sulfate. For example, 50% inhibition of the FtsZ assembly and GTP hydrolysis occurred at 0.1 and 0.2 m of urea, respectively. FtsZ lost its functional properties before any significant perturbation in the secondary or tertiary structure was detected by using several fluorescence techniques and far UV-CD indicating preferential local unfolding of the functional region(s). In addition, the unfolded FtsZ regains its ability to polymerize fully upon removal of urea. The data taken together suggest that FtsZ unfolds reversibly through a multistep process, and local responses that inhibit functional properties precede the global transition of FtsZ to the unfolded state.     

Elicitation of simian immunodeficiency virus-specific cytotoxic T lymphocytes in mucosal compartments of rhesus monkeys by systemic vaccination

Since most human immunodeficiency virus (HIV) infections are initiated following mucosal exposure to the virus, the anatomic containment or abortion of an HIV infection is likely to require vaccine-elicited cellular immune responses in those mucosal sites. Studying vaccine-elicited mucosal immune responses has been problematic because of the difficulties associated with sampling T lymphocytes from those anatomic compartments. In the present study, we demonstrate that mucosal cytotoxic T lymphocytes (CTL) specific for simian immunodeficiency virus (SIV) and simian HIV can be reproducibly sampled from intestinal mucosal tissue of rhesus monkeys obtained under endoscopic guidance. These lymphocytes recognize peptide-major histocompatibility complex class I complexes and express gamma interferon on exposure to peptide antigen. Interestingly, systemic immunization of monkeys with plasmid DNA immunogens followed by live recombinant attenuated poxviruses or adenoviruses with genes deleted elicits high-frequency SIV-specific CTL responses in these mucosal tissues. These studies therefore suggest that systemic delivery of potent HIV immunogens may suffice to elicit substantial mucosal CTL responses.     

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.