The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0PE, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0PE has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery. © 2012 Elsevier Inc.

Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.

Magnitude differences in bioactive compounds, chemical functional groups, fatty acid profiles, nutrient degradation and digestion, molecular structure and metabolic characteristics of protein in newly developed yellow-seeded and black-seeded canola lines.

Recently, new lines of yellow-seeded (CS-Y) and black-seeded canola (CS-B) have been developed with chemical and structural alteration through modern breeding technology. However, no systematic study was found on the bioactive compounds, chemical functional groups, fatty acid profiles, inherent structure, nutrient degradation and absorption, or metabolic characteristics between the newly developed yellow- and black-seeded canola lines. This study aimed to systematically characterize chemical, structural, and nutritional features in these canola lines. The parameters accessed include bioactive compounds and antinutrition factors, chemical functional groups, detailed chemical and nutrient profiles, energy value, nutrient fractions, protein structure, degradation kinetics, intestinal digestion, true intestinal protein supply, and feed milk value. The results showed that the CS-Y line was lower (P ≤ 0.05) in neutral detergent fiber (122 vs 154 g/kg DM), acid detergent fiber (61 vs 99 g/kg DM), lignin (58 vs 77 g/kg DM), nonprotein nitrogen (56 vs 68 g/kg DM), and acid detergent insoluble protein (11 vs 35 g/kg DM) than the CS-B line. There was no difference in fatty acid profiles except C20:1 eicosenoic acid content (omega-9) which was in lower in the CS-Y line (P < 0.05) compared to the CS-B line. The glucosinolate compounds differed (P < 0.05) in terms of 4-pentenyl, phenylethyl, 3-CH3-indolyl, and 3-butenyl glucosinolates (2.9 vs 1.0 μmol/g) between the CS-Y and CS-B lines. For bioactive compounds, total polyphenols tended to be different (6.3 vs 7.2 g/kg DM), but there were no differences in erucic acid and condensed tannins with averages of 0.3 and 3.1 g/kg DM, respectively. When protein was portioned into five subfractions, significant differences were found in PA, PB1 (65 vs 79 g/kg CP), PB2, and PC fractions (10 vs 33 g/kg CP), indicating protein degradation and supply to small intestine differed between two new lines. In terms of protein structure spectral profile, there were no significant differences in functional groups of amides I and II, α helix, and β-sheet structure as well as their ratio between the two new lines, indicating no difference in protein structure makeup and conformation between the two lines. In terms of energy values, there were significant differences in total digestible nutrient (TDN; 149 vs 133 g/kg DM), metabolizable energy (ME; 58 vs 52 MJ/kg DM), and net energy for lactation (NEL; 42 vs 37 MJ/kg DM) between CS-Y and CS-B lines. For in situ rumen degradation kinetics, the two lines differed in soluble fraction (S; 284 vs 341 g/kg CP), potential degradation fraction (D; 672 vs 590 g/kg CP), and effective degraded organic matter (EDOM; 710 vs 684 g/kg OM), but no difference in degradation rate. CS-Y had higher digestibility of rumen bypass protein in the intestine than CS-B (566 vs 446 g/kg of RUP, P < 0.05). Modeling nutrient supply results showed that microbial protein synthesis (MCP; 148 vs 171 g/kg DM) and rumen protein degraded balance (DPB; 108 vs 127 g/kg DM) were lower in the CS-Y line, but there were no differences in total truly digested protein in small intestine (DVE) and feed milk value (FMV) between the two lines. In conclusion, the new yellow line had different nutritional, chemical, and structural features compared to the black line. CS-Y provided better nutrient utilization and availability.

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

Ruminal dry matter and nitrogen degradation in relation to condensed tannin and protein molecular structures in sainfoin (Onobrychis viciifolia) and lucerne (Medicago sativa).

Sainfoin is a temperate legume that contains condensed tannins (CT), i.e. polyphenols that are able to bind proteins and thus reduce protein degradation in the rumen. A reduction in protein degradation in the rumen can lead to a subsequent increase in amino acid flow to the small intestine. The effects of CT in the rumen and the intestine differ according to the amount and structure of CT and the nature of the protein molecular structure. The objective of the present study was to investigate the degradability in the rumen of three CT-containing sainfoin varieties and CT-free lucerne in relation to CT content and structure (mean degree of polymerization, proportion of prodelphinidins and cis-flavanol units) and protein structure (amide I and II bands, ratio of amide I-to-amide II, α-helix, β-sheet, ratio of α-helix-to-β-sheet). Protein molecular structures were identified using Fourier transform/infrared-attenuated total reflectance (FT/IR-ATR) spectroscopy. The in situ degradability of three sainfoin varieties (Ambra, Esparcette and Villahoz) was studied in 2008, during the first growth cycle at two harvest dates (P1 and P2, i.e. 5 May and 2 June, respectively) and at one date (P3) during the second growth cycle (2 June) and these were compared with a tannin-free legume, lucerne (Aubigny). Loss of dry matter (DMDeg) and nitrogen (NDeg) in polyester bags suspended in the rumen was measured using rumen-fistulated cows. The NDeg of lucerne compared with sainfoin was 0·80 v. 0·77 at P1, 0·78 v. 0·65 at P2 and 0·79 v. 0·70 at P3, respectively. NDeg was related to the rapidly disappearing fraction (‘a’) fraction (r=0·76), the rate of degradation (‘c’) (r=0·92), to the content (r=−0·81) and structure of CT. However, the relationship between NDeg and the slowly disappearing fraction (‘b’) was weak. There was a significant effect of date and species×date, for NDeg and ‘a’ fraction. The secondary protein structure varied with harvest date (species×date) and was correlated with the fraction ‘b’. Both tannin and protein structures influenced the NDeg degradation. CT content and structure were correlated to the ‘a’ fraction and to the ‘c’. Features of the protein molecular secondary structure were correlated to the ‘b’ fraction.

Role of different proteolytic pathways in degradation of muscle protein from streptozotocin-diabetic rats

In vitro rates of overall proteolysis and the activities of four different proteolytic pathways (lysosomal, Ca2+ dependent, ATP dependent, and ATP independent), as well as rates of protein synthesis, were measured in soleus and extensor digitorum longus (EDL) muscles from streptozotocin- diabetic rats. In the acute phase (1-3 days) of diabetes, there was an increase in overall proteolysis that coincided with an increased activity of the Ca2+-dependent pathway in both soleus and EDL and of the ATP-dependent pathway in EDL. After longer periods (5-10 days) of diabetes, the overall rate of protein degradation decreased and reached values similar to or even lower than those of controls as a result of a reduction in the activities of Ca2+-dependent and ATP-dependent pathways. No change was detected at any time interval in the activity of the intralysosomal proteolytic system in muscles from diabetic animals. Rates of protein synthesis were already reduced 24 h after diabetes induction and decreased further thereafter. Insulin treatment restored to normal the activities of the proteolytic pathways and rates of protein synthesis.

Lysosomal degradation of RhoH protein upon antigen receptor activation in T but not B cells

RhoH is a member of the Rho (ras homologous) GTPase family, yet it lacks GTPase activity and thus remains in its active conformation. Unlike other Rho GTPases, the RhoH gene transcript is restricted to hematopoietic cells and RhoH was shown to be required for adequate T-cell activation through the TCR. Here, we demonstrate that both blood T and B cells, but not neutrophils or monocytes, express RhoH protein under physiological conditions. Upon TCR complex activation, RhoH was degraded in lysosomes of primary and Jurkat T cells. Pharmacologic activation of T cells distal to the TCR complex had no effect on RhoH protein levels suggesting that early events during T-cell activation are required for RhoH protein degradation. In contrast to T cells, activation of the BCR in blood B cells was not associated with changes in RhoH levels. These data suggest that RhoH function might be regulated by lysosomal degradation of RhoH protein following TCR complex but not BCR activation. This newly discovered regulatory pathway of RhoH expression might limit TCR signaling and subsequent T-cell activation upon Ag contact.

Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein

The bacterial iron response regulator (Irr) protein mediates iron-dependent regulation of heme biosynthesis. Pulse–chase and immunoprecipitation experiments showed that Irr degraded in response to 6 μM iron with a half-life of ≈30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron. Addition of heme to low iron (0.3 μM) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum of heme to a shorter wavelength. A Cys29 → Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

In vivo examination of membrane protein localization and degradation with green fluorescent protein.

To test the utility of green fluorescent protein (GFP) as an in vivo reporter protein when fused to a membrane domain, we made a fusion protein between yeast hydroxymethylglutaryl-CoA reductase and GFP. Fusion proteins displayed spatial localization and regulated degradation consistent with the native hydroxymethylglutaryl-CoA reductase proteins. Thus, GFP should be useful in the study of both membrane protein localization and protein degradation in vivo.

Skeletal muscle atrophy, a link between depression of protein synthesis and increase in degradation

Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2α). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2α phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRΔ6. Myotubes expressing PKRΔ6 showed no increase in eIF2α phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-κB (NF-κB). Inhibition of PKR prevented nuclear migration of NF-κB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-κB. Phosphorylation of PKR and eIF2α was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

Effect of a tumour-produced lipid-mobilizing factor on protein synthesis and degradation

Treatment of murine myoblasts, myotubes and tumour cells with a tumour-produced lipid mobilizing factor (LMF), caused a concentration-dependent stimulation of protein synthesis, within a 24 h period. There was no effect on cell number or [3H] thymidine incorporation, but a similar concentration-dependent stimulation of 2-deoxyglucose uptake. LMF produced an increase in intracellular cyclic AMP levels, which was linearly (r2 = 0.973) related to the increase in protein synthesis. The effect of LMF was attenuated by the adenylate cyclase inhibitor MDL12330A, and was additive with the stimulation produced by forskolin. Both propranolol (10 μM) and the specific β3-adrenergic receptor antagonist SR 59230A (10-5M), significantly reduced the stimulation of protein synthesis induced by LMF. Protein synthesis was also increased by 69% (P = 0.006) in soleus muscles of mice administered LMF, while there was a 26% decrease in protein degradation (P = 0.03). While LMF had no effect on the lysosomal enzymes, cathepsins B and L, there was a decrease in proteasome activity, as determined both by the 'chymotrypsin-like' enzyme activity, as well as expression of proteasome α-type subunits, determined by Western blotting. These results show that in addition to its lipid-mobilizing activity LMF also increases protein accumulation in skeletal muscle both by an increase in protein synthesis and a decrease in protein catabolism. © 2001 Cancer Research Campaign.

Cholesterol enhances phospholipid-dependent activated protein C anticoagulant activity

The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities. © 2005 International Society on Thrombosis and Haemostasis.

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of doublestranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5′ end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 59 thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.