Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2

<p>The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C₂H₂-type zinc-finger, and only the C₂H₂-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C₂H₂-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations.</p

Structural basis of FYCO1 and MAP1LC3A interaction reveals a novel binding mode for Atg8-family proteins

<p>FYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general.</p

Facile Solvothermal Synthesis of Phase-Pure Cu₄O₃ Microspheres and Their Lithium Storage Properties

Phase-pure Cu₄O₃ microspheres were synthesized for the first time via a facile solvothermal method, using Cu­(NO₃)₂·3H₂O as the precursor. A formation mechanism was proposed based on the observation of a series of reaction intermediates. The samples were characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, temperature-programmed reduction and oxidation, X-ray photoelectron spectroscopy, and nitrogen adsorption. It was found that the composition of the prepared products were highly dependent on the synthesis conditions, particularly the hydrate water content in the copper precursor of Cu­(NO₃)₂. Pure Cu₄O₃ microspheres with a diameter of 2–10 μm could be obtained via the symproportionation reaction (2CuO + Cu₂O → Cu₄O₃), which was regarded not being feasible in aqueous media under mild synthesis conditions. The electrochemical properties of the Cu₄O₃ microspheres as anode materials for Li-ion batteries were also investigated. Compared to the simple physical mixture of CuO and Cu₂O with an equivalent atomic ratio of 2:1, the as-prepared Cu₄O₃ exhibited unique lithium storage behaviors at a low voltage range and superior electrochemical performances as an anode material for Li-ion batteries. The successful preparation of pure Cu₄O₃ material could provide opportunities to further explore its physicochemical properties and potential applications

Radiographic analyses of mice reveal multiple subcutaneous ossifications.

<p>X-rays of 12 month +/−p and WT mice. A) 12 month +/−p female with no ossifications visualized; B) 12 month +/−p male, inset and arrows demonstrate areas consistent with ossifications; C) 12 month WT without areas of ossifications.</p

The dermis and subcutis of 3 month old male mice with heterozygous targeted disruption of exon 1 of the <i>Gnas</i> gene.

<p>The −m/+ and +/−p male and female mice had no heterotopic bone formation. However, there were subtle lesions in the dermis of both −m/+ and +/−p male mice that comprised widely scattered plaque-like areas with pale collagen and increased cellularity in periadnexal areas of the reticular dermis. A) −m/+ male; B) +/−p male; Scale bar = 50 µm.</p

The KEN box is essential for BRSK2 degradation.

<p>The same amount of wild type or various mutant HA-BRSK2 expression constructs were co-transfected with (–) or without (+) Myc-Cdh1 or Myc-Cdc20 plasmid into HeLa cells. The protein levels of BRSK2 and Cdh1 were determined by Western blotting (WB) with anti-HA and anti-Myc antibodies, respectively. The pEGFP-C1 expression construct was included as a control for transfection efficiency.</p

The ossifications appear to originate in the perifollicular areas.

<p>Ossifications are emanating near the hair follicle both with hematoxylin and eosin staining (A) as well as Alizarin Red staining (B). Scale bar = 50 µm.</p

The APC/C co-activator Cdh1 promotes BRSK2 degradation.

<p>(A) Sequence alignment of BRSK2 orthologs from different species, including <i>Homo sapiens</i>, <i>Mus musculus, Gallus gallus</i> and <i>Bos taurus</i>. The two conserved D boxes and the KEN box are outlined. (B) HA-BRSK2 was co-transfected with increasing amounts of Myc-Cdh1 or Myc-Cdc20 into HeLa cells. MG132 (20 µM) was added 6 h before harvest. Cells were harvested, and exogenous BRSK2 protein levels were analyzed. (C) Increasing amounts of Myc-Cdh1 were transfected into HeLa cells that were treated with MG132 similarly to (B). Cells were harvested, and endogenous BRSK2 protein levels were analyzed. pEGFP-C1 co-transfection in (B) and (C) served as a control for transfection efficiency, and GFP levels were assessed using anti-GFP antibodies. (D) HeLa cells were transiently transfected with one or both of two Cdh1-specific siRNAs or control siRNA. Forty-eight hours after transfection, BRSK2 and Cdh1 protein levels were analyzed by Western blotting (WB). (E) Expression of Cdh1 was knocked down by co-transfection of the two Cdh1-specific siRNAs. HeLa cells were treated with 10 ng/µL cycloheximide for the indicated times. HeLa cells treated for 6 h were also treated with (+) or without (−) MG132. Whole-cell extracts were Western blotted (WB) with the indicated antibodies. The graph shows the quantification of BRSK2 levels using actin as a control. The error bars represent the mean ± S.D. from three independent experiments. (F) HEK293T cells were arrested by a nocodazole block and lysed, and then, the cell lysates were incubated with Protein A/G-Sepharose conjugated with either control IgG or Cdh1 antibody. The immunoprecipitates were washed, and bound proteins were resolved on an SDS-PAGE gel followed by Western blotting (WB).</p

BRSK2 stability is controlled by the ubiquitin-proteasome pathway.

<p>(A) HEK293T cells were treated with each 20 µM MG132 or 10 µM lactacystin for the indicated times. Equal amounts of total cell lysates were subjected to Western blot (WB) analysis using antibodies against BRSK2, Cyclin B1 and β-actin. The mean values (± S.D.) of three independent experiments are shown. (B) qRT-PCR measurements of BRSK2 mRNA levels in HEK293T cells after treatment with lactacystin at the indicated time points. GAPDH mRNA levels were used for normalization. (C) HEK293T cells were transfected with (–) or without (+) HA-BRSK2 or Flag-Ub as indicated. BRSK2 was immunoprecipitated (IP) with anti-HA antibody, and the immunocomplexes were analyzed by SDS-PAGE and Western blotting (WB) analysis using anti-HA and anti-Flag M2 antibodies.</p

Over-expression of BRSK2 increases the population of cells in G2/M.

<p>Control, BRSK2(WT) or BRSK2(ΔKEN) plasmids were transfected with pBB14-GFP plasmid into HeLa cells and H1299 cells. Cells were harvested for flow cytometry analysis and Western blotting (WB) analysis using indicated antibody. The pBB14-GFP expression construct served as a positive transfection marker for flow cytometry analysis.</p