Structural dissection of a gating mechanism preventing misactivation of ubiquitin by NEDD8's E1

Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL's C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin's Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8's heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin's E1 UBA1, is a key UBL selectivity determinant. Here, we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8's residue 72 and UBA3's residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3's preference for NEDD8's Ala72 appears to be indirect, due to proper positioning of UBA3's Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin's Arg72 and an E1's Gln. However, APPBP1-UBA3's failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3's Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.     

The alpha-N-acetyl-glucosaminidase gene is transcriptionally activated in male and female gametes prior to fertilization and is essential for seed development in Arabidopsis

Sugar residues in proteoglycan complexes carry important signalling and regulatory functions in biology. In humans, heparan sulphate is an example of such a complex polymer containing glucosamine and N-acetyl-glucosamine residues and is present in the extracellular matrix. Although heparan sulphate has not been found in plants, the At5g13690 gene encoding the alpha-N-acetyl-glucosaminidase (NAGLU), an enzyme involved in its catabolism, is present in the Arabidopsis genome. Among our collection of embryo-defective lines, a plant was identified in which the T-DNA had inserted into the AtNAGLU gene. The phenotype of atnaglu is an early arrest of seed development without apparent male or female gametophytic effects. These data demonstrated the essential function in Arabidopsis consistent with the contribution of NAGLU to the Sanfilippo syndrome in human. Expression of AtNAGLU in plants was shown to be prevalent during reproductive development. The presence of AtNAGLU mRNA was observed during early and late male gametogenesis and in each cell of the embryo sac at the time of fertilization. After fertilization, AtNAGLU was expressed in the embryo, suspensor, and endosperm until the cotyledonary stage embryo. This precise pattern of expression identifies the cells and tissues where a remodelling of the N-acetyl-glucosamine residues of proteoglycan complexes is occurring. This work provides original evidence of the important role of N-acetyl-glucosamines in plant reproductive development.     

A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.     

Modulation of the tumor cell phenotype by IFN-gamma results in resistance of uveal melanoma cells to granule-mediated lysis by cytotoxic lymphocytes

IFN-gamma, a pleiotropic immune regulator, is implicated in both tumor immune surveillance and selection of tumor variants resistant to immune control, i.e., immunoediting. In uveal melanoma patients, elevated serum levels of IFN-gamma correlate with the spread of metastasis and represent a negative prognostic marker. Treatment with IFN-gamma boosted the MHC class I presentation machinery in uveal melanoma cells but suppressed their MHC class I-restricted CTL lysis. Tumor cells exposed to IFN-gamma efficiently activated specific CTL but were less susceptible to permeabilization by perforin and exhibited a decreased capacity to bind and incorporate granzyme B. These results define a novel mechanism of resistance to granule-mediated CTL lysis in human tumors. Furthermore, the data suggest that immunoediting is not limited to genetic or epigenetic changes resulting in stable cellular phenotypes but also involves an inducible modulation of tumor cells in response to a microenvironment associated with immune activation.     

Cyclic AMP-dependent protein kinase controls energy interconversion during the catalytic cycle of the yeast copper-ATPase

The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase-mediated phosphorylation. However, there is no evidence showing the influence of kinase-related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP-dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)-ATPase, with PKA-mediated phosphorylation of a conserved serine (Ser258) being crucial for catalysis. Long-range intramolecular communication between Ser258 and Asp627 (at the catalytic site) modulates the key pumping event: the conversion of the high-energy to the low-energy phosphorylated intermediate associated with copper release.     

A note on the time budget and social behaviour of densely housed horses: A case study in Arab breeding mares

We observed a high-density herd (200 mares/ha) of 44 Arab breeding mares, while in a bare paddock in Tunisia. Twenty-minute animal focal samples and scan sampling were used to determine the time budget of the mares during the period from 9 a.m. to 3 p.m. and study their social behaviour. The data obtained reveal restricted behavioural repertoires with missing behaviour like rolling, allogrooming and lying down; unusual time budgets with a high frequency of locomotion that constitutes the most frequent activity (27.9 ± 19.47%) of the mares. Social interactions were restricted to agonistic interactions but despite the high stocking density, aggressions were not that frequent among mares.     

Use of Low-Molecular�CWeight Heparin to Decrease Mortality in Mice after Intracardiac Injection of Tumor Cells

Intracardiac injection of human tumor cells into anesthetized nude mice is an established model of bone metastasis. However, intracardiac injection of some human tumor cell lines cause acute neurologic signs and high mortality, making some potentially relevant tumor cell lines unusable for investigation. We showed that intracardiac injection of tumor cells can induce a hypercoagulable state leading to platelet consumption and thromboemboli formation and that pretreatment with intravenous injection of low-molecular–weight heparin (LMWH; enoxaparin) blocks this state. In addition, intravenous injection of enoxaparin before intracardiac injection with 2 different small-cell lung carcinoma lines, H1975 and H2126, dramatically decreased mouse mortality while still generating bone metastases. Therefore, reduction of mortality by pretreatment with LMWH increases the types of cells that can be studied in this metastasis model and decreases the number of animals used.Abbreviations: APTT, activated partial thromboplastin time; BLI, bioluminescent imaging; CBC, complete blood count; DIC, disseminated intravascular coagulation; H1975luc, H1975 cell line tagged with lucerifase–green fluorescent protein; H2126luc, H2126 cell line tagged with lucerifase–green fluorescent protein; LMWH, low-molecular–weight heparin; PT, prothrombin time; UFH, unfractionated heparinResearch using animal models mimicking the metastasis of human tumors to bone is critical for the development of cancer therapeutics. Bone metastases are present in almost all people who die of cancer and are more likely to occur with breast, prostate, lung, kidney, and thyroid cancers.^1,24 In patients with advanced breast and prostate cancers, much of the tumor burden at the time of death will be found in bone.²⁰ The pattern of bone metastases can range from purely destructive (osteolytic) to mostly osteoblastic (bone-forming) lesions. Osteolysis is accompanied by pain, bone fragility, and increased susceptibility to pathologic fracture. In osteolytic metastasis, a 2-way interaction between tumor cells and osteoclasts in the bone microenvironment leading to continued osteolysis and tumor growth is suspected.²⁰ Current therapies for bone metastases, such as bisphosphonates, are directed at inhibiting bone resorption, but other therapies are in development that specifically target tumor cell or osteoclast factors involved in the 2-way cycle between tumor growth and osteolysis.¹⁸Bone metastasis is rare in mouse models of spontaneous mammary and prostate carcinomas, experimentally implanted animal tumor models (such as syngeneic and xenograft tumors), and chemical or transgenic induction of mammary and prostate carcinomas. To increase the frequency of bone metastases, injection techniques using either orthotopic tumor cell injection into mammary glands or prostate or intracardiac injection of human tumor cell lines into the left ventricle of nude mice have been developed.^5,14,25,31 In contrast to the late stage, low incidence of metastasis after orthotopic injection, intracardiac injection of human tumor cell lines results in much higher rates of bone metastasis at an early stage in the disease, with osteolytic metastases to the metaphyses of long bones.^6,23 Development of osteolytic lesions in this model can be monitored by various methods, including radiography and, more recently, in vivo bioluminescent imaging (BLI) using lucerifase-tagged tumor cells. Bioluminescent imaging detects micrometastatic lesions and allows for serial in vivo monitoring of bone metastases.^9-11 After a BLI study, bone metastases can be assessed histologically, with tumor foci typically seen in the femur or tibia.Bone metastasis models using the intracardiac tumor injection technique have been primarily focused on a few breast (for example, MDA-MB-231) and prostate models (for example, PC3), but additional models of other tumors that interact with bone (especially lung carcinomas) need to be developed.^24,30 Intracardiac injection of some nonsmall cell lung carcinoma tumor cell lines have led to stroke-like clinical signs, including head tilt, spinning, and failure to recover from anesthesia after intracardiac injection.¹⁵ We postulated that the stroke-like clinical signs and mortality were due to thromboembolism formation immediately after intracardiac tumor cell injection.Tumor cells have procoagulant activity. Procoagulants, such as tissue factor, may be increased on the surface of or secreted into the blood by cancer cells, leading to changes in the clotting cascade.¹³ Approximately 15% of all cancer patients are affected by thromboembolic disease, including superficial and deep-vein thrombosis, arterial thrombosis and embolism, pulmonary emboli, and thrombosis of venous access devices.^12,13 Anticoagulant treatments used clinically to prevent thrombi and thromboemboli include warfarin, unfractionated heparin (UFH), and low-molecular–weight heparins (LMWH), such as enoxaparin (Lovenox, Sanofi Aventis, Bridgewater, NJ) and dalteparin (Fragmin, Pfizer, New York, NY). LMWHs are prepared through chemical, hydrolytic, or enzymatic degradation of unfractionated heparin.¹³ Both UFH and LMWH exert their anticoagulant effects by binding to antithrombin and causing a confrontational change. This change increases the interaction of antithrombin with thrombin (IIa) and activated factors X (Xa) and IX (IXa), leading to inhibition of clotting.^8,28LMWHs decrease the formation of thromboembolism and subsequent mortality in several murine models of thromboembolism and disseminated intravascular coagulation (DIC). In the murine model of thrombin-induced thromboembolism, massive deposition of intravascular fibrin—mainly within the pulmonary arteries—causes death within 5 minutes after thrombin injection.^16,22 Both UFH and LMWH inhibit thrombin and prevent mortality in this model, but bleeding times and activated partial prothrombin time (APPT) are less prolonged with LMWH.¹⁶ LMWH is also effective in preventing murine DIC in a lipopolysaccharide model, in which mice given 2 injections of lipopolysaccharide develop DIC, multiple organ failure, and die. Mice given LMWH before lipopolysaccharide administration have fewer lung and liver microthrombi and greater survival than do mice not given LMWH.^26,27Here, we evaluated the use of LMWH in mice to prevent morbidity and mortality associated with intracardiac injection of human tumor cell lines. We determined that thromboembolism occurred in intracardiac tumor-challenged mice and that LMWH blocked thromboembolism. We also determined the effect of LMWH on animal survival and subsequent development of bone metastasis in this mouse model.     

Relief of microRNA-mediated translational repression in human cells subjected to stress

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.