RNF185, a novel mitochondrial ubiquitin E3 ligase, regulates autophagy through interaction with BNIP1.

Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy.

[Expression of NKG2D and NKG2A with their ligands MHC-I A/B and HLA-E in acute leukemia patients and its significance].

This study was aimed to explore the difference of NK cell receptor NKG2D and NKG2A expression on NK cells and CD3(+) T cells and their ligand MHC-I A/B (major histocompatibility complex class I-related chains A/B) and HLA-E expression in leukemia cells, as well as its immunological significance. Flow cytometry was used to detect the killing rate of NK92 cells to 8 leukemia cell lines, and the expression of NKG2D and NKG2A on NK cells and CD3(+) T cells as well as their ligand MHC-I A/B and HLA-E expression on leukemia cells. The results indicated that the NK92 showed different killing activity to different leukemia cell lines. The positive expression rate of NKG2D and NKG2A on NK cells and CD3(+) T cells in ALL patients was no significantly different from that in AML patients (p > 0.05), but positive expression rate of MHC-I A/B and HLA-E in ALL patients was obviously higher than that in AML patients (p < 0.05). It is concluded that there is difference of immune cell function between ALL and AML patients, this difference may be associated with the expression difference of NKG2D and NKG2A ligands on leukemia cells while does not associated with the killing and inhibiting receptors expressed on NK cells and CD3(+) T cells.

RNF152, a novel lysosome localized E3 ligase with pro-apoptotic activities.

RING finger protein 152 (RNF152) is a novel RING finger protein and has not been well characterized. We report here that RNF152 is a canonical RING finger protein and has E3 ligase activity. It is polyubiqitinated partly through Lys-48-linked ubiquitin chains in vivo and this phenomenon is dependent on its RING finger domain and transmembrane domain. RNF152 is localized in lysosomes and co-localized with LAMP3, a lysosome marker. Moreover, over-expression of RNF152 in Hela cells induces apoptosis. These results suggest that RNF152 is a lysosome localized E3 ligase with pro-apoptotic activities. It is the first E3 ligase identified so far that is involved in lysosome-related apoptosis.

Tumor necrosis factor-alpha blockade leads to decreased peripheral T cell reactivity and increased dendritic cell number in peripheral blood of patients with ankylosing spondylitis.

OBJECTIVE: To study the effect of tumor necrosis factor-alpha (TNF-alpha) antagonist (etanercept) treatment on the peripheral T cell reactivity of patients with ankylosing spondylitis (AS). METHODS: Peripheral blood mononuclear cells were collected from 40 patients with AS at baseline, after 2 and 6 weeks of etanercept treatment or placebo treatment, and from healthy controls. The number of cells secreting various cytokines was detected by enzyme linked immunospot. Serum soluble interleukin 2 (IL-2) receptor level was measured by ELISA. T cell proliferation was assayed with the WST-1 live cell-staining method. The myeloid dendritic cell (mDC) and regulatory T cell (Treg) levels were analyzed by fluorescence activated cell sorting. RESULTS: . After 2 and 6 weeks of etanercept treatment, the number of TNF-alpha-secreting monocytes decreased. Although the T cell proliferation rate remained stable, the number of T cells secreting IL-2 and interferon-gamma under anti-CD3/anti-CD28 stimulation was significantly decreased. The level of serum soluble IL-2R (sIL-2R), a T cell activation marker, also declined. The changes in T cell reactivity were correlated with a significant increase in MHC Class II-positive mDC cells in circulation. An increase in Treg cell numbers was also observed. CONCLUSION: . The anti-TNF-alpha therapy blockaded MHC Class II-positive mDC maturation, enhanced regulatory T cell levels, and suppressed the functions of effector T cells. The reduced T cell reactivity could contribute to the efficacy of the TNF-alpha antagonist therapy in patients with AS.

A novel protein tyrosine kinase NOK that shares homology with platelet- derived growth factor/fibroblast growth factor receptors induces tumorigenesis and metastasis in nude mice.

Receptor protein tyrosine kinases (RPTKs) play important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Here, we report the identification and characterization of a novel RPTK-like molecule that has a critical role in induction of tumorigenesis and metastasis and is termed Novel Oncogene with Kinase-domain (NOK). NOK contains a putative single transmembrane domain and a conserved intracellular tyrosine kinase domain that shares homology with members of the platelet-derived growth factor/fibroblast growth factor receptor superfamily. NOK was exclusively located in the cytoplasm. NOK mRNAs were detected in limited human organs and expressed with the highest abundance in the prostate. A variety of tumor cells also expressed the NOK mRNAs. We demonstrated that NIH3T3 and BaF3 cells could be strongly transformed by the expression of the NOK gene as examined by colony formation experiment. In addition, BaF3 cells with the stable expression of NOK induced rapid tumorigenesis in nude mice. Interestingly, these NOK-expressing tumor cells could promptly invade and spread into various distinct organs and form metastatic foci, eventually leading to the rapid death of these animals. Moreover, molecular mechanism studies indicated that NOK could concomitantly activate both MAP kinase and phosphatidylinositol 3'-kinases (PI3K) pathways in stable BaF3 cells. Thus, our results both in vitro and in vivo suggest that NOK is a novel oncogene with the capacity of promoting cell transformation, tumorigenesis, and metastasis.

Synthesis and potential antimetastatic activity of monovalent and divalent beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-D-glucopyranosides.

Anomers of monovalent and divalent beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-D-gluco-pyranosides were synthesized under different glycosylation conditions, and evaluated for in vitro antimetastatic activity. Three compounds showed promising inhibitory effects on cancer cell attachment, spreading, migration, and invasion.