Description: Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells.

Description: Mouse pluripotent cells, such as embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), provide excellent in vitro systems to study imperative pre- and postimplantation events of in vivo mammalian development. It is known that mouse ESCs are dynamic heterogeneous populations. However, it remains largely unclear whether and how EpiSCs possess heterogeneity and plasticity similar to that of ESCs. Here, we show that EpiSCs are discriminated by the expression of a specific marker T (Brachyury) into two populations. The T-positive (T+) and the T-negative (T−) populations can be interconverted within the same culture condition. In addition, the two populations display distinct responses to bone morphogenetic protein (BMP) signaling and different developmental potentials. The T− EpiSCs are preferentially differentiated into ectoderm lineages, whereas T+ EpiSCs have a biased potential for mesendoderm fates. Mechanistic studies reveal that T+ EpiSCs have an earlier and faster response to BMP4 stimulation than T− EpiSCs. Id1 mediates the commitment of T− EpiSCs to epidermal lineage during BMP4 treatment. On the other hand, Snail modulates the conversion of T+ EpiSCs to mesendoderm fates with the presence of BMP4. Furthermore, T expression is essential for epithelial-mesenchymal transition during EpiSCs differentiation. Our findings suggest that the dynamic heterogeneity of the T+/T− subpopulation primes EpiSCs toward particular cell lineages, providing important insights into the dynamic development of the early mouse embryo.

Description: Naturally occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic N-glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivity toward the residues around the Asn of N-glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10) N-glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.

Description: The spike protein N-terminal domains (NTDs) of bovine coronavirus (BCoV) and mouse hepatitis coronavirus (MHV) recognize sugar and protein receptors, respectively, despite their significant sequence homology. We recently determined the crystal structure of MHV NTD complexed with its protein receptor murine carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which surprisingly revealed a human galectin (galactose-binding lectin) fold in MHV NTD. Here, we have determined at 1.55 Å resolution the crystal structure of BCoV NTD, which also has the human galectin fold. Using mutagenesis, we have located the sugar-binding site in BCoV NTD, which overlaps with the galactose-binding site in human galectins. Using a glycan array screen, we have identified 5-N-acetyl-9-O-acetylneuraminic acid as the preferred sugar substrate for BCoV NTD. Subtle structural differences between BCoV and MHV NTDs, primarily involving different conformations of receptor-binding loops, explain why BCoV NTD does not bind CEACAM1 and why MHV NTD does not bind sugar. These results suggest a successful viral evolution strategy in which coronaviruses stole a galectin from hosts, incorporated it into their spike protein, and evolved it into viral receptor-binding domains with altered sugar specificity in contemporary BCoV or novel protein specificity in contemporary MHV.

Description: The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase functions as a central node in the DNA damage response signaling network. The mechanisms by which ATR activity is amplified and/or maintained are not understood. Here we demonstrate that BRIT1/microcephalin (MCPH1), a human disease-related protein, is dispensable for the initiation but essential for the amplification of ATR signaling. BRIT1 interacts with and recruits topoisomerase-binding protein 1 (TopBP1), a key activator of ATR signaling, to the sites of DNA damage. Notably, replication stress-induced ataxia telangiectasia-mutated or ATR-dependent BRIT1 phosphorylation at Ser-322 facilitates efficient TopBP1 recruitment. These results reveal a mechanism that ensures the continuation of ATR-initiated DNA damage signaling. Our study uncovers a previously unknown regulatory axis of ATR signaling in maintaining genomic integrity, which may provide mechanistic insights into the perturbation of ATR signaling in human diseases such as neurodevelopmental defects and cancer.

Description: Sustained therapeutic hypercapnia prevents pulmonary hypertension in experimental animals, but its rescue effects on established disease have not been studied. Therapies that inhibit Rho-kinase (ROCK) and/or augment nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling can reverse or prevent progression of chronic pulmonary hypertension. Our objective in the present study was to determine whether sustained rescue treatment with inhaled CO 2 (therapeutic hypercapnia) would improve structural and functional changes of chronic hypoxic pulmonary hypertension. Spontaneously breathing pups were exposed to normoxia (21% O 2 ) or hypoxia (13% O 2 ) from postnatal days 1–21 with or without 7% CO 2 (Pa CO 2 elevated by ~25 mmHg) or 10% CO 2 (Pa CO 2 elevated by ~40 mmHg) from days 14 to 21 . Compared with hypoxia alone, animals exposed to hypoxia and 10% CO 2 had significantly ( P 〈 0.05) decreased pulmonary vascular resistance, right-ventricular systolic pressure, right-ventricular hypertrophy, and medial wall thickness of pulmonary resistance arteries as well as decreased lung phosphodiesterase (PDE) V, RhoA, and ROCK activity. Rescue treatment with 10% CO 2 , or treatment with a ROCK inhibitor (15 mg/kg ip Y-27632 twice daily from days 14 to 21 ), also increased pulmonary arterial endothelial nitric oxide synthase and lung NO content. In contrast, cGMP content and cGMP-dependent protein kinase (PKG) activity were increased by exposure to 10% CO 2 , but not by ROCK inhibition with Y-27632. In vitro exposure of pulmonary artery smooth muscle cells to hypercapnia suppressed serum-induced ROCK activity, which was prevented by inhibition of PKG with Rp-8-Br-PET-cGMPS. We conclude that sustained hypercapnia dose-dependently inhibited ROCK activity, augmented NO-cGMP-PKG signaling, and led to partial improvements in the hemodynamic and structural abnormalities of chronic hypoxic PHT in juvenile rats. Increased PKG content and activity appears to play a major upstream role in CO 2 -induced suppression of ROCK activity in pulmonary arterial smooth muscle.