An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes.

A close chromatin conformation precludes gene expression in eukaryotic cells. Genes activated by external cues have to overcome this repressive state by locally changing chromatin structure to a more open state. Although much is known about hormonal gene activation, how basal repression of regulated genes is targeted to the correct sites throughout the genome is not well understood. Here we report that in breast cancer cells, the unliganded progesterone receptor (PR) binds genomic sites and targets a repressive complex containing HP1γ (heterochromatin protein 1γ), LSD1 (lysine-specific demethylase 1), HDAC1/2, CoREST (corepressor for REST [RE1 {neuronal repressor element 1} silencing transcription factor]), KDM5B, and the RNA SRA (steroid receptor RNA activator) to 20% of hormone-inducible genes, keeping these genes silenced prior to hormone treatment. The complex is anchored via binding of HP1γ to H3K9me3 (histone H3 tails trimethylated on Lys 9). SRA interacts with PR, HP1γ, and LSD1, and its depletion compromises the loading of the repressive complex to target chromatin-promoting aberrant gene derepression. Upon hormonal treatment, the HP1γ-LSD1 complex is displaced from these constitutively poorly expressed genes as a result of rapid phosphorylation of histone H3 at Ser 10 mediated by MSK1, which is recruited to the target sites by the activated PR. Displacement of the repressive complex enables the loading of coactivators needed for chromatin remodeling and activation of this set of genes, including genes involved in apoptosis and cell proliferation. These results highlight the importance of the unliganded PR in hormonal regulation of breast cancer cells.

Danshen (Salvia miltiorrhiza) restricts MD2/TLR4-MyD88 complex formation and signalling in acute myocardial infarction-induced heart failure.

Heart failure (HF) represents a major public health burden. Inflammation has been shown to be a critical factor in the progression of HF, regardless of the aetiology. Disappointingly, the majority of clinical trials targeting aspects of inflammation in patients with HF have been largely negative. Many clinical researches demonstrate that danshen has a good efficacy on HF, and however, whether danshen exerts anti-inflammatory effects against HF remains unclear. In our study, the employment of a water extracted and alcohol precipitated of danshen extract attenuated cardiac dysfunction and inflammation response in acute myocardial infarction-induced HF rats. Transcriptome technique and validation results revealed that TLR4 signalling pathway was involved in the anti-inflammation effects of danshen. In vitro, danshen reduced the release of inflammatory mediators in LPS-stimulated RAW264.7 macrophage cells. Besides, the LPS-stimulated macrophage conditioned media was applied to induce cardiac H9C2 cells injury, which could be attenuated by danshen. Furtherly, knock-down and overexpression of TLR4 were utilized to confirm that danshen ameliorated inflammatory injury via MyD88-dependent TLR4-TRAF6-NF-κB signalling pathway in cardiomyocytes. Furthermore, by utilizing co-immunoprecipitation, danshen was proved to suppress MD2/TLR4 complex formation and MyD88 recruitment. In conclusion, our results demonstrated that danshen ameliorates inflammatory injury by controlling MD2/TLR4-MyD88 complex formation and TLR4-TRAF6-NF-κB signalling pathway in acute myocardial infarction-induced HF.

A De novo Peptide from a High Throughput Peptide Library Blocks Myosin A -MTIP Complex Formation in Plasmodium falciparum.

Apicomplexan parasites, through their motor machinery, produce the required propulsive force critical for host cell-entry. The conserved components of this so-called glideosome machinery are myosin A and myosin A Tail Interacting Protein (MTIP). MTIP tethers myosin A to the inner membrane complex of the parasite through 20 amino acid-long C-terminal end of myosin A that makes direct contacts with MTIP, allowing the invasion of Plasmodium falciparum in erythrocytes. Here, we discovered through screening a peptide library, a de-novo peptide ZA1 that binds the myosin A tail domain. We demonstrated that ZA1 bound strongly to myosin A tail and was able to disrupt the native myosin A tail MTIP complex both in vitro and in vivo. We then showed that a shortened peptide derived from ZA1, named ZA1S, was able to bind myosin A and block parasite invasion. Overall, our study identified a novel anti-malarial peptide that could be used in combination with other antimalarials for blocking the invasion of Plasmodium falciparum.

Rabex-5 is a lenalidomide target molecule that negatively regulates TLR-induced type 1 IFN production.

Immunomodulatory drugs (IMiDs) are a family of compounds derived from thalidomide. Binding of the IMiD molecule to the Lon protease Cereblon initiates the degradation of substrates via the ubiquitin proteasome pathway. Here, we show that Cereblon forms a complex with Rabex-5, a regulator of immune homeostasis. Treatment with lenalidomide prevented the association of Cereblon with Rabex-5. Conversely, mutation of the IMiD binding site increased Cereblon-Rabex-5 coimmunoprecipitation. The thalidomide binding region of Cereblon therefore regulates the formation of this complex. Knockdown of Rabex-5 in the THP-1 macrophage cell line up-regulated Toll-like receptor (TLR)-induced cytokine and type 1 IFN production via a STAT1/IRF activating pathway. Thus, we identify Rabex-5 as a IMiD target molecule that functions to restrain TLR activated auto-immune promoting pathways. We propose that release of Rabex-5 from complex with Cereblon enables the suppression of immune responses, contributing to the antiinflammatory properties of IMiDs.

Effects of Long-Term Alcohol Drinking on the Dopamine D2 Receptor: Gene Expression and Heteroreceptor Complexes in the Striatum in Rats.

BACKGROUND: Reduced dopamine D2 receptor (D2R) ligand binding has repeatedly been demonstrated in the striatum of humans with alcohol use disorder (AUD). The attenuated D2R binding has been suggested to reflect a reduced D2R density, which in turn has been proposed to drive craving and relapse. However, results from rodent studies addressing the effects of alcohol drinking on D2R density have been inconsistent. METHODS: A validated alcohol drinking model (intermittent access to 20% alcohol) in Wistar rats was used to study the effects of voluntary alcohol drinking (at least 12 weeks) on the D2R in the striatum compared to age-matched alcohol-naïve control rats. Reverse transcriptase quantitative PCR was used to quantify isoform-specific Drd2 gene expression levels. Using bisulfite pyrosequencing, DNA methylation levels of a regulatory region of the Drd2 gene were determined. In situ proximity ligation assay was used to measure densities of D2R receptor complexes: D2R-D2R, adenosine A2A receptor (A2AR)-D2R, and sigma1 receptor (sigma1R)-D2R. RESULTS: Long-term voluntary alcohol drinking significantly reduced mRNA levels of the long D2R isoform in the nucleus accumbens (NAc) but did not alter CpG methylation levels in the analyzed sequence of the Drd2 gene. Alcohol drinking also reduced the striatal density of D2R-D2R homoreceptor complexes, increased the density of A2AR-D2R heteroreceptor complexes in the NAc shell and the dorsal striatum, and decreased the density of sigma1R-D2R heteroreceptor complexes in the dorsal striatum. CONCLUSIONS: The present results on long-term alcohol drinking might reflect reduced D2R levels through reductions in D2R-D2R homoreceptor complexes and gene expression. Furthermore, based on antagonistic interactions between A2AR and D2R, an increased density of A2AR-D2R heteroreceptor complexes might indicate a reduced affinity and signaling of the D2R population within the complex. Hence, both reduced striatal D2R levels and reduced D2R protomer affinity within the striatal A2AR-D2R complex might underlie reduced D2R radioligand binding in humans with AUD. This supports the hypothesis of a hypodopaminergic system in AUD and suggests the A2AR-D2R heteroreceptor complex as a potential novel treatment target.

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.

Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies.

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility.

The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients.

Dopamine D2 receptors gate generalization of conditioned threat responses through mTORC1 signaling in the extended amygdala.

Overgeneralization of conditioned threat responses is a robust clinical marker of anxiety disorders. In overgeneralization, responses that are appropriate to threat-predicting cues are evoked by perceptually similar safety-predicting cues. Inappropriate learning of conditioned threat responses may thus form an etiological basis for anxiety disorders. The role of dopamine (DA) in memory encoding is well established. Indeed by signaling salience and valence, DA is thought to facilitate discriminative learning between stimuli representing safety or threat. However, the neuroanatomical and biochemical substrates through which DA modulates overgeneralization of threat responses remain poorly understood. Here we report that the modulation of DA D2 receptor (D2R) signaling bidirectionally regulates the consolidation of fear responses. While the blockade of D2R induces generalized threat responses, its stimulation facilitates discriminative learning between stimuli representing safety or threat. Moreover, we show that controlled threat generalization requires the coordinated activation of D2R in the bed nucleus of the stria terminalis and the central amygdala. Finally, we identify the mTORC1 cascade activation as an important molecular event by which D2R mediates its effects. These data reveal that D2R signaling in the extended amygdala constitutes an important checkpoint through which DA participates in the control of threat processing and the emergence of overgeneralized threat responses.

Hydrophobic Interactions Are Key To Drive the Association of Tapasin with Peptide Transporter Subunit TAP2.

The transporter associated with Ag processing (TAP) translocates proteasomally derived cytosolic peptides into the endoplasmic reticulum. TAP is a central component of the peptide-loading complex (PLC), to which tapasin (TPN) recruits MHC class I (MHC I) and accessory chaperones. The PLC functions to facilitate and optimize MHC I-mediated Ag presentation. The heterodimeric peptide transporter consists of two homologous subunits, TAP1 and TAP2, each of which contains an N-terminal domain (N-domain) in addition to a conserved transmembrane (TM) core segment. Each N-domain binds to the TM region of a single TPN molecule, which recruits one MHC I molecule to TAP1 and/or TAP2. Although both N-domains act as TPN-docking sites, various studies suggest a functional asymmetry within the PLC resulting in greater significance of the TAP2/TPN interaction for MHC loading. In this study, we demonstrate that the leucine-rich hydrophobic sequence stretches (with the central leucine residues L20 and L66) in the first and second TM helix of TAP2 form a functional unit acting as a docking site for optimal TPN/MHC I recruitment, whereas three distinct highly conserved arginine and/or aspartate residues inside or flanking these TM helices are dispensable. Moreover, we show that the physical interaction between TAP2 and TPN is disrupted by benzene, a compound known to interfere with hydrophobic interactions, such as those between pairing leucine zippers. No such effects were observed for the TAP1/TAP2 interaction or the complex formation between TPN and MHC I. We propose that TAP/TPN complex formation is driven by hydrophobic interactions via leucine zipper-like motifs.

Effect of Acute Alcohol Ingestion on Resistance Exercise-Induced mTORC1 Signaling in Human Muscle.

Duplanty, AA, Budnar, RG, Luk, HY, Levitt, DE, Hill, DW, McFarlin, BK, Huggett, DB, and Vingren, JL. Effect of acute alcohol ingestion on resistance exercise-induced mTORC1 signaling in human muscle. J Strength Cond Res 31(1): 54-61, 2017-The purpose of this project was to further elucidate the effects postexercise alcohol ingestion. This project had many novel aspects including using a resistance exercise (RE) only exercise design and the inclusion of women. Ten resistance-trained males and 9 resistance-trained females completed 2 identical acute heavy RE trials (6 sets of Smith machine squats) followed by ingestion of either alcohol or placebo. All participants completed both conditions. Before exercise (PRE) and 3 (+3 hours) and 5 (+5 hours) hours postexercise, muscle tissue samples were obtained from the vastus lateralis by biopsies. Muscle samples were analyzed for phosphorylated mTOR, S6K1, and 4E-BP1. For men, there was a significant interaction effect for mTOR and S6K1 phosphorylation. At +3 hours, mTOR and S6K1 phosphorylation was higher for placebo than for alcohol. For women, there was a significant main effect for time. mTOR phosphorylation was higher at +3 hours than at PRE and at +5 hours. There were no significant effects found for 4E-BP1 phosphorylation in men or women. The major findings of this study was that although RE elicited similar mTORC1 signaling both in men and in women, alcohol ingestion seemed to only attenuate RE-induced phosphorylation of the mTORC1 signaling pathway in men. This study provides evidence that alcohol should not be ingested after RE as this ingestion could potentially hamper the desired muscular adaptations to RE by reducing anabolic signaling, at least in men.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 µmol·kg-1·h-1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

Targeting of mTORC2 may have advantages over selective targeting of mTORC1 in the treatment of malignant pheochromocytoma.

Recent studies have found that mammalian target of rapamycin complex 2 (mTORC2) is emerging as a potential therapeutic target in the treatment of many human cancers. However, the effects of targeting of mTORC2 on malignant pheochromocytomas (PCC) and paragangliomas (PGL) have not been reported. The aim of the study was to investigate the effects of targeting of mTORC2 on malignant PCC/PGL by comparing the inhibitory effects of targeting of mTORC2 with mTORC1 on pheochromocytoma PC12 cell in vitro and vivo. The expressions of regulatory-associated protein of mTOR (raptor) and rapamycin-insensitive companion of mTOR (rictor) were detected by immunohistochemistry in human tissues of malignant PCC. Targeting of mTORC1, mTORC2, and mTORC1/2 (mTORC1 and mTORC2) were performed by transfected with raptor, rictor, and mammalian target of rapamycin (mTOR) small interfering RNA (siRNA) in pheochromocytoma PC12 cell, respectively. MTT assay, apoptosis analysis, wound healing, and Transwell approach were performed. A tumor model in nude mice bearing PC12 cell xenografts, which were dosed with rapamycin or PP242, was established. The expression of raptor was frequently moderate positive, but the expression of rictor was frequently strong positive in malignant PCC. In vitro, although inhibition of mTORC1 was able to suppress PC12 cell proliferation, inhibition of mTORC2 more effectively suppressed cell proliferation. Inhibition of mTORC2 or mTORC1/2 more effectively prevented cell migration and invasion, and promoted cell apoptosis, while inhibition of mTORC1 only slightly prevented cell migration and invasion, and was not able to promoted apoptosis. Also, we found that mTOR downstream kinases were deregulated by targeting of mTORC2, but not mTORC1. In vivo, we found that PP242 was more potent than rapamycin in inhibiting tumor growth in tumor model. Our data suggest that targeting of mTORC2 may have advantages over selective targeting of mTORC1 in the treatment of malignant PCC/PGL. However, more clinical trials are needed to prove our findings.

Alcohol intoxication following muscle contraction in mice decreases muscle protein synthesis but not mTOR signal transduction.

BACKGROUND: Alcohol (ethanol [EtOH]) intoxication antagonizes stimulation of muscle protein synthesis and mammalian target of rapamycin (mTOR) signaling. However, whether the anabolic response can be reversed when alcohol is consumed after the stimulus is unknown. METHODS: A single bout of electrically stimulated muscle contractions (10 sets of 6 contractions) was induced in fasted male C57BL/6 mice 2 hours prior to alcohol intoxication. EtOH was injected intraperitoneally (3 g/kg), and the gastrocnemius/plantaris muscle complex was collected 2 hours later from the stimulated and contralateral unstimulated control leg. RESULTS: Muscle contraction increased protein synthesis 28% in control mice, while EtOH abolished this stimulation-induced increase. Further, EtOH suppressed the rate of synthesis ~75% compared to control muscle irrespective of stimulation. This decrease was associated with impaired protein elongation as EtOH increased the phosphorylation of eEF2 Thr(56) . In contrast, stimulation-induced increases in mTOR protein complex-1 (mTORC1) (S6K1 Thr(421) /Ser(424) , S6K1 Thr(389) , rpS6 Ser(240/244) , and 4E-BP1 Thr(37/46) ) and mitogen-activated protein kinase (MAPK) (JNK Thr(183) /Tyr(185) , p38 Thr(180) /Tyr(182) , and rpS6S(235/236) ) signaling were not reversed by acute EtOH. CONCLUSIONS: These data suggest that EtOH-induced decreases in protein synthesis in fasted mice may be independent of mTORC1 and MAPK signaling following muscle contraction and instead due to the antagonistic actions of EtOH on mRNA translation elongation. Therefore, EtOH suppresses the contraction-induced increase in protein synthesis, and over time has the potential to prevent skeletal muscle hypertrophy induced by repeated muscle contraction.

Inhibition by stabilization: targeting the Plasmodium falciparum aldolase-TRAP complex.

BACKGROUND: Emerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the 'glideosome', an actin-myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals. METHODS: Virtual library screening of a large compound library against the PfAldolase-TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods. RESULTS: A novel inhibitory effect on the parasite was achieved by stabilizing a protein-protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase-TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization. CONCLUSION: This proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein-protein interaction within the parasite, an avenue towards inhibiting a previously "undruggable" target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95 % in the presence of the small molecule stabilizer compound 24.

Increased mammalian target of rapamycin complex 2 signaling promotes age-related decline in CD4 T cell signaling and function.

CD4 T cell function declines significantly during aging. Although the mammalian target of rapamycin (TOR) has been implicated in aging, the roles of the TOR complexes (TORC1, TORC2) in the functional declines of CD4 T cells remain unknown. In this study, we demonstrate that aging increases TORC2 signaling in murine CD4 T cells, a change blocked by long-term exposure to rapamycin, suggesting that functional defects may be the result of enhanced TORC2 function. Using overexpression of Rheb to activate TORC1 and Rictor plus Sin1 to augment TORC2 in naive CD4 T cells from young mice, we demonstrated that increased TORC2, but not TORC1, signaling results in aging-associated biochemical changes. Furthermore, elevated TORC2 signaling in naive CD4 T cells from young mice leads to in vivo functional declines. The data presented in this article suggest a novel model in which aging increases TORC2 signaling and leads to CD4 T cell defects in old mice.

Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.

Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5ß1, αVß1, αVß3 and αVß6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVß3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-ß1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5ß1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the ß subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.

2P2Idb: a structural database dedicated to orthosteric modulation of protein-protein interactions.

Protein-protein interactions are considered as one of the next generation of therapeutic targets. Specific tools thus need to be developed to tackle this challenging chemical space. In an effort to derive some common principles from recent successes, we have built 2P2Idb (freely accessible at http://2p2idb.cnrs-mrs.fr), a hand-curated structural database dedicated to protein-protein interactions with known orthosteric modulators. It includes all interactions for which both the protein-protein and protein-ligand complexes have been structurally characterized. A web server provides links to related sites of interest, binding affinity data, pre-calculated structural information about protein-protein interfaces and 3D interactive views through java applets. Comparison of interfaces in 2P2Idb to those of representative datasets of heterodimeric complexes has led to the identification of geometrical parameters and residue properties to assess the druggability of protein-protein complexes. A tool is proposed to calculate a series of biophysical and geometrical parameters that characterize protein-protein interfaces. A large range of descriptors are computed including, buried accessible surface area, gap volume, non-bonded contacts, hydrogen-bonds, atom and residue composition, number of segments and secondary structure contribution. All together the 2P2I database represents a structural source of information for scientists from academic institutions or pharmaceutical industries.

Chronic rapamycin treatment exacerbates metabolism and does not down-regulate mTORC2/Akt signaling in diabetic mice induced by high-fat diet and streptozotocin.

Rapamycin, a classical inhibitor of the mammalian target of rapamycin (mTOR), has been intensively studied for its role in metabolism and verified to induce metabolic defects through mTORC2/Akt pathway. However, disparity of the results exists depending on the differences of the animal models or the detailed procedures. Moreover, data regarding the effect of rapamycin treatment in diabetic models are sparse. Therefore, we investigated its influence on glucose and lipid metabolism, and further analyzed its effect on the mTORC2/Akt pathway in a high-fat diet- and streptozotocin-induced diabetic mice model. Three-weeks old C57BL/6J mice were fed with a high fat diet (60 kcal% fat) and intraperitoneally injected with streptozotocin (100 mg/kg) at 6 weeks of age. Rapamycin (2 mg/kg) was orally given to the mice daily for consecutive 6 weeks. Body weight, blood lipid parameters and HbA(1c)% values were evaluated. Oral glucose test and insulin tolerance test were performed. Furthermore, western blot assay was applied to investigate the protein epression levels of Akt and PKCα, two key targets of the mTORC2/Akt pathway. Rapamycin-treated diabetic mice demonstrated less weight gain, more profound symptoms of polydipsia, polyphagia and polyuria, significant liver fat accumulation and exacerbated metabolic disorders including insulin resistance, hyperglycemia and dyslipidemia. Contrary to what have been expected, though significantly inhibiting mTORC1/S6K1 signaling, chronic rapamycin treatment failed to down-regulate mTORC2/Akt pathway. Our findings provide evidence that chronic rapamycin treatment may exacerbate metabolism in diabetic subjects and does not down-regulate mTORC2/Akt signialing in a high-fat diet- and streptozotocin-induced diabetic mice model.