The many factors influencing the shear wave velocity (SWV) measured with Acoustic Radiation Force Impulse (ARFI) are examined in order to define the most correct examination technique. In particular, attention is given to the information achieved by experimental models, such as phantoms and animal studies. This review targets the clinical applications of ARFI in the evaluation of chronic diffuse disease, especially of liver and kidneys. The contribution of ARFI to the clinical workout of these patients and some possible perspectives are described.Stiffness significantly varies among normal and abnormal biological tissues.• In clinical applications physical, geometrical, anatomical and physiological factors influence the SWV.• Elastographic techniques can quantify fibrosis, which is directly related to stiffness.• ARFI can be useful in chronic diffuse disease of liver and kidney.•  Adopted from clinical practice from long ago, palpation examines the mechanical properties of target organs following the general logic shared by all modern imaging methods: it explores tissues detecting the effects determined by an external force. While conventional radiology and computed tomography extract information from the differential attenuation of X-photons due to density and to the mean atomic number in the body volume sampled, the background of palpation-based diagnosis lies in the relationship linking several diseases to the modifications of tissue stiffness that they induce. The principle of palpation has been recently applied to a series of elasticity-based modalities generally defined as “strain-imaging techniques”, in which diagnostic information derives from the response of the explored tissue (closely related to its elastic properties) to forces inducing mechanical modifications. Both quasi-static  and dynamic strain-imaging techniques have been developed: these latter use as a stimulus rapidly attenuating shear waves resulting from mechanical vibrations. Such vibrations can be applied either outside (like in Transient Elastography: TE) or in “internal” techniques, directly inside the body.Acoustic Radiation Force Impulse (ARFI) is a recently developed, dynamic, internal technique that superimposes data concerning tissue elasticity to a conventional gray-scale image generated by commercial ultrasound (US) scanners. In this article, the basic technological principles of the ARFI method, with emphasis on the factors that determine the results, are reported, and its current diagnostic role in diffuse disease  is critically reviewed.In the ARFI technique, the shear waves exciting the target tissue are generated inside a fixed-size 1 x 0.5 cm) region of interest (ROI), placed at the choice of the operator on a conventional gray-scale ultrasound (US) image. In brief, focused, short-duration acoustic push pulses travelling along the main US beam  x 0.5 cm. In turnTogether with the push pulses, low-intensity tracking US beams are continuously emitted parallel to the main beam in order to monitor the tissue displacement. The tracking beams intercept the shear wave front at several predetermined locations and time intervals, which allows a series of data concerning the tissue response to be obtained, such as the time-to-peak displacement and the recovery time Fig. . From thPhysical factors affecting the propagation of the shear waves are both intrinsic to the main US beam and extrinsic. The most relevant intrinsic factor is the wavelength: if lower transmitting frequencies are used, lower-frequency pulses are generated, which in turn exert greater acoustic pressure, resulting in faster-travelling shear waves. Exploring both experimental phantoms and normal tissues in vivo with different US frequencies , a slighAmong the physical factors extrinsic to the US beam, two variables have to be considered. The stronger the compression manually exerted on the transducer, the higher the tissue density becomes, which increases the speed of propagation of the shear waves Fig. ; in an eGeometry too plays a role in determining the speed of propagation of the shear waves. The more parallel the ROI is oriented to the main US beam, the higher the SWV results are in a given target Fig. , presumaAt least two categories of anatomical factors strongly influence the propagation of the shear waves. As a first consideration, within some organs—composed of macroscopic portions each having its own structure—uneven obstacles are opposed to the progression of the shear waves, similarly to the differences in the acoustic impedance regulating the transmission of US beams. Shear waves are expected to meet more architectural disturbances in the sinus than in the cortex of normal kidneys, which is the likely basis of the lower sinusal SWV values Fig. .Fig. 7EfThe most important anatomical factor, however, is anisotropy, which reflects the direction dependence of certain properties that some organs demonstrate Fig. . Most ofA marked anisotropy is normally present in the renal medulla, where—within a given renal segment—Henle loops and vasa recta are parallel to each other, all being oriented from the capsule to the papilla Fig. ; the samLike anisotropy, the physiological factors affecting the propagation of the shear waves are especially relevant in the kidneys. In the above-mentioned model , a lineaUnder ideal experimental conditions , the elasticity of the target volume is the only determinant of the SWV values measured by ARFI Fig. . In clinPrior to the validation of its diagnostic performances, ARFI had to be demonstrated to be a safe technique, quick and easy to perform and providing reproducible results in different settings.The range of energy of the acoustic beams is similar in common ARFI practice  and in conventional gray-scale US. At these levels, the absorption of acoustic energy in biological tissue is expected—besides acoustic radiation force—to generate heat, the amount of which is mainly influenced by transmit frequency and pulse duration. For an individual excitation, the temperature rise is very limited, ranging from 0.02 to 0.2 °C: it takes less energy to displace tissue several microns than to raise its temperature by a fraction of a degree Celsius .Although radiation forces generated by US beams having intensities and frequencies commonly used in conventional US can cause tissue displacement until a maximum depth shallower than the corresponding B-mode imaging depth , from thThe disturbing effect of motion artifacts, caused by both transducer and underlying physiological movements, is easily removed by motion filters; furthermore, multiple reference tracking beams are emitted before push pulses are generated in order to sample baseline motion .As a subsequent historical step in the validation of ARFI technique, images were achieved with a good correspondence to conventional US images, both on cancers after their surgical removal and on normal organs in vivo , especiaWith regard to the evaluation of normal and abnormal kidneys, however, a widespread acceptance of the reproducibility of the results of ARFI has not been achieved to date , 23–26. Chronic liver disease is very common in clinical practice: its more frequent causes are infection with hepatitis viruses B and C, ethanol abuse, non-alcoholic steatohepatitis, autoimmune hepatitis, and primary biliary cirrhosis. In these patients, a precise estimation of liver fibrosis is crucial both for the planning of treatment  and for the assessment of outcome, the degree of fibrosis representing the strongest prognostic indicator –30. On aOn the assumption that a direct proportionality exists between the degree of fibrosis and liver stiffness, both quasi-static and dynamic shear-wave elastographic techniques have been widely used, aimed at a quick, non-invasive and reliable quantification of fibrosis. Among the latter group, TE was introduced in clinical practice more than a decade ago. Several meta-analyses assessed that the measurement of liver stiffness by means of TE, in patients with chronic hepatopathies, significantly correlates with the histological degree of fibrosis –35, alloFor historical reasons, therefore, this clinical application of ARFI has been initially validated through a comparison with the already known diagnostic performances of TE, and in no correlative study were significant differences demonstrated between the accuracies of ARFI and TE , 36–42. p < 0.0001), even if a large overlap of values occurred in the stages of 2 or below, with only F ≥ 2 and cirrhosis being excluded with great certainty [For the assessment of liver fibrosis Fig. , ARFI scertainty . Better ertainty , 56, andertainty .Fig. 10AAs for chronic liver disease due to less common etiologies, ARFI proved reliable in differentiating significant from non-significant steatosis in children with biliary atresia  and in aThe accuracy of ARFI in the prediction of complications of chronic liver disease is more controversial. Although in most cases the risk of bleeding from esophageal varices is low, the current guidelines recommend endoscopy in all patients, in order to identify those who would benefit from prophylactic treatment , which l99mTc-DMSA), a procedure associated with a certain amount of radiation exposure, is recommended by the current guidelines in the evaluation of several pediatric forms, especially in European countries [An extension of the excellent results obtained by ARFI in the liver to the evaluation of kidneys could be foreseen on the basis that a hypercellular, high-grade interstitial fibrosis leading to a progressive loss of functioning nephrons is the common final histological pathway of chronic kidney disease, whatever its cause , and alsountries , 69. A pountries  showed tountries .However, uneven results have been reported concerning the reliability of ARFI in the evaluation of renal allografts. According to Syversveen , ARFI caIn chronic kidney disease of the adult, one author  did not As for the pediatric population, in one study in which children younger than 2 years with pelvis dilation were examined, SWV values were significantly higher than in normal controls only when high-grade hydronephrosis was present . In our In acute pancreatitis, SWV values are reported to be significantly higher than in normal pancreas. One group obtained 97 % sensitivity and 93 % specificity in differentiating patients with acute pancreatitis from both normal subjects and patients with chronic pancreatitis using a 2.2 m/s SWV cutoff value ; in anotIn one study, SWV values were significantly higher in patients with chronic pancreatitis than in healthy volunteers Fig. : the senFew other studies regarding the ARFI evaluation of diffuse pancreatic disease can be found. In patients with cystic fibrosis, SWV values were lower when pancreatic insufficiency existed . A higheWhen the stiffness of surgical thyroid specimens was measured, a significant direct relationship of SWV values to the histological degree of fibrosis (regardless of the underlying disease) emerged . As a coARFI-based quantification of tissue stiffness is a non-invasive, cheap, safe and quick imaging tool potentially capable of improving the accuracy of US examinations. ARFI reliably quantifies the degree of fibrosis in chronic liver disease, contributing to treatment planning and prognosis assessment, and can probably help in the staging of diffuse chronic disease of other organs, especially the kidneys. Given the high number of factors significantly influencing the measurements, the importance of a rigorous, target-tailored examination technique is critical in order to obtain reliable results.


Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described ‘spatial rheostat’ controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin one and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs.DOI:http://dx.doi.org/10.7554/eLife.23172.001 In animal bodies, a mesh of proteins – known the extracellular matrix – holds cells together to give the body shape and make it more stable. Cells bind to the matrix using structures called focal adhesions. However, cells do not always stay in one place: in young animals, certain cells need to move around the body to reach their final destination. Adult animals also have some cells that are able to move, for example, to close wounds. The cells move when the focal adhesions that hold these cells in place are taken apart and then rebuilt. These processes are very dynamic and happen all the time when cells move. They are normally tightly controlled to ensure that cells only migrate under appropriate conditions. However, focal adhesions are less well regulated in cancer cells, allowing the cells to migrate away from a tumour to form new tumours elsewhere in the body.Focal adhesions are large structures that contain many proteins. These proteins include FAK and Src, which are particularly important and have been well studied. In order to better understand how focal adhesions are taken apart, Schoenherr et al. wanted to discover new proteins that interact with FAK in skin cancer cells from mice. The experiments show that FAK binds to a protein called Ambra1, which is known to control how other proteins move around inside cells. Ambra1 and FAK work together to regulate the movement of Src away from focal adhesions and into the cell. Furthermore, Ambra1 belongs to a larger network of proteins within the cancer cells that regulates the location of Src. By changing the levels of Src and FAK at focal adhesions, Ambra1 and its other binding partners can control whether the cancer cells are attached to the extracellular matrix or are free to migrate.Overall this work shows that the location and activity of Src within cells needs to be carefully controlled to stop the cells from moving at the wrong time. Further experiments will aim to understand which other proteins are involved in this network and how they contribute to the growth of cancer cells and the spread of tumours around the body.DOI:http://dx.doi.org/10.7554/eLife.23172.002 We therefore investigated the role of Ambra1 in tumour-associated phenotypes regulated by the Src/FAK pathway in squamous cell carcinoma (SCC) cells derived from the DMBA/TPA model of carcinogenesis (driven by mutated oncogenic H-Ras) . We haveWe found that Ambra1 is critically involved in Src/FAK-dependent cancer cell polarisation and chemotactic invasion. In FAK-depleted SCC cells, Ambra1 controls the targeting of active Src to intracellular autophagic puncta. This is mediated by the novel Ambra1-binding proteins Dynactin 1  and interferon-induced transmembrane protein 3 (IFITM3), which together with Ambra1, are critical to trafficking processes that control spatial Src activity and Src-dependent phenotypes. A FAK mutant that is not able to bind Ambra1 promotes increased cell adhesion and invasion by retaining more phospho-FAK (pFAK) and phospho-Src (pSrc) at focal adhesions – showing that Ambra1 can both positively and negatively regulate the amount of active Src at cellular adhesion sites. Overall, these data imply a novel role for the autophagic protein Ambra1, and its key interacting partners Dynactin 1 and IFITM3, at the heart of an intracellular trafficking network that, in turn, acts as a ‘spatial rheostat’ for phospho-Src and Src/FAK-mediated cancer processes.We discovered Ambra1 in a phage display screen for novel FAK binding partners. As Ambra1 regulates autophagy, and we had already published that active Src was trafficked away from adhesions via autophagosomes in the absence of FAK , we set The IPs were validated using FAK-deficient (-/-) SCC cells and Ambra1 -/- mouse embryonic fibroblasts (MEFs) . In ordeWe previously showed that active Src is localised to intracellular puncta containing autophagy proteins in FAK -/- SCC cells, allowing FAK-deficient cancer cells to cope with high levels of ‘untethered’ active Src  , 2012b. To investigate whether Ambra1 controlled active Src localisation, we suppressed expression of Ambra1 with both a pool of four and two individual siRNAs . KnockdoWe next performed Ambra1, pSrc Y416, and FAK (and IgG control) IPs using lysates from FAK-WT and FAK -/- cells (in triplicate) and determined specifically interacting proteins by quantitative label-free mass spectrometry. Amongst the binding partners of all three ‘baits’ were proteins that are involved in regulating intracellular trafficking, raising the possibility that Ambra1 is part of a Src/FAK ‘trafficking network’ –3.10.755To test the hypothesis that Ambra1-dependent targeting of pSrc from focal adhesions to intracellular autophagic puncta involved such trafficking proteins, we selected two novel Ambra1 binding partners for further study. These were Dynactin 1, whose interaction with Ambra1 appeared to be enriched in FAK-deficient cells from proteomics analyses  and IFITTo determine whether IFITM3 was required for complex formation between active Src and Ambra1 in FAK-WT and -/- cells, we knocked down IFITM3 using a pool of siRNAs and analysed the interaction of Ambra1 and pSrc by co-immunoprecipitations . While kvia interaction with proteins involved in intracellular trafficking, including Dynactin 1 and IFITM3; the latter lies at the centre of linked Ambra1, pSrc, and FAK interactomes.Having verified that both Dynactin 1 and IFITM3 bind to Ambra1, we next addressed whether they were involved in the Ambra1-dependent intracellular trafficking of active pSrc. We transiently knocked down endogenous Dynactin 1  or IFITMHaving established that Ambra1 and FAK interact with each other and co-localise at focal adhesions , and thaAs Ambra1 binds FAK and is found at isolated focal adhesions, we next addressed whether FAK and Ambra1 interact directly. The Ambra1 binding site in FAK was mapped by peptide array binding analysis as described previously , which rWhen culturing the cells expressing FAK that was impaired in Ambra1 binding, we noticed that these FAK P875A/P881A cells seemed to display greater adherence than the FAK-WT cells. Therefore, we performed adhesion assays on fibronectin-coated dishes  or on plTo determine whether there were other biological consequences of the aberrant accumulation, or retention, of active pSrc and pFAK at focal adhesions when FAK binding to Ambra1 was impaired, we examined chemotactic invasion into growth factor-reduced Matrigel and proliferation in three-dimensional (3D) culture. We found that elevated active pSrc and pFAK at focal adhesions in FAK P875A/P881A cells was associated with enhanced invasive migration  and incrOur results define Ambra1 as a crucial regulator of Src/FAK-mediated cancer phenotypes, acting as a ‘spatial rheostat’ to maintain steady-state levels of active pSrc and pFAK at adhesions  included the dynactin complex component Dynactin 1 and IFITM3, in addition to other proteins considered to be intracellular trafficking proteins. As Ambra1 has been reported to interact with the dynein complex, we thought it likely that Ambra1 may regulate the trafficking of active Src to intracellular autophagic puncta rocesses , and we rocesses , implyinrocesses , and losrocesses . Knockdorocesses  that plaExpression of a mutant FAK protein (in otherwise FAK-deficient cancer cells) that was hugely impaired in its ability to bind Ambra1 (FAK P875A/P881A), demonstrated the importance of the complex between FAK and Ambra1 in promoting the removal of phosphorylated components from focal adhesions, including active pSrc and pFAK. This Ambra1 binding-impaired mutant FAK protein caused increased steady-state levels of active pSrc and pFAK at focal adhesions, and enhanced adhesion, proliferation in 3D and chemotactic invasive migration, without affecting Src/FAK binding. Enhanced invasion was reversed by knockdown of the Ambra1-binding trafficking proteins Dynactin 1 and IFITM3, demonstrating that these Ambra1-scaffolded proteins are involved in the dynamic regulation of active pSrc and pFAK, presumably by trafficking them to and from focal adhesions. Indeed, we found that FAK-driven invasion in the SCC cells was hugely dependent on IFITM3, implying that its crucial role at the heart of trafficking protein networks linking Ambra1, FAK and active Src is vital to Src/FAK-mediated invasion. One caveat of interpreting the data with the FAK P875A/P881A mutant protein is that this mutant is also impaired in binding p130Cas, although to a much lesser extent than Ambra1; importantly though, and in contrast to knockdown of Ambra1, knockdown of p130Cas did not significantly affect invasion (not shown).In summary, we have identified a novel role for Ambra1 as part of an intracellular trafficking network of proteins that control the steady-state levels, and dynamics, of active Src and FAK at focal adhesions. Ambra1 scaffolds proteins such as Dynactin 1 and IFITM3 – and here, for the first time, we link these to the spatial control of active Src and FAK, regulating their steady-state levels at focal adhesions and the autophagic targeting of active Src when FAK is absent. As a result, Ambra1 and its interacting partners control cancer cell adhesion, polarisation, proliferation in 3D and chemotactic invasion. Deregulation of these trafficking components of focal adhesion complexes inhibits Src/FAK-dependent biological processes in cancer cells. Our work demonstrates the importance of tight dynamic control of trafficking of active Src, in particular, to and from focal adhesions.AB_647289), anti-GM130 (RRID:AB_398141), anti-p130Cas (RRID:AB_397667) and anti-RACK1 (RRID:AB_397577) antibodies , anti-IFITM3 (RRID:AB_2122095) , anti-CoxIV (RRID:AB_10694213), anti-FAK (RRID:AB_10694068), anti-pFAK Y397 (RRID:AB_2173659), anti-pPaxillin Y118 (RRID:AB_2174466), anti-Rab7 (RRID:AB_1904103), anti-pSrc Y416 (RRID:AB_331697), anti-Src (clone 36D10) (RRID:AB_10693939), anti-LC3B (RRID:AB_2137707) and anti-GAPDH (RRID:AB_10622025) , and anti-Dctn1 (RRID:AB_10842517), anti-Ambra1 (RRID:AB_2636939) and anti-pSrc Y416 (RRID:AB_309898) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID:AB_310789) . LC3B antibody was from MBL (RRID:AB_1279144) . TRITC-phalloidin was purchased from Life Technologies (RRID:AB_2572408) . Anti-rabbit (RRID:AB_2099233) or mouse (RRID:AB_330924) peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb  and PF562271 from Pfizer .Antibodies used were as follows: anti-Paxillin  and P881A . After DpnI digestion for 1 hr at 37°C, chemically competent TOP10 bacteria were transformed.FAK mutants were generated by site-directed mutagenesis using PFU Ultra Hotstart DNA polymerase  and the following primers (mutated base pairs are underlined): P875A  and were negative for all pathogens tested. All cell lines were routinely tested negative for mycoplasma contamination.FAK-deficient SCC cell lines were generated, authenticated and characterised as described previously , 2010. S Velasco . MEFs weAmbra1 siRNA pool (cat. no. M-059556–01), individual Ambra1 siRNAs , Dctn1 siRNA pool (cat. no. M-044821–01), IFITM3 siRNA pool (cat. no. M-056653–01) or scrambled siRNA (cat. no. D-001206-13-20) were purchased from Dharmacon . FAK-WT or FAK -/- SCC cells were transiently transfected using HiPerFect  according to the manufacturer’s protocol, with a final concentration of 100 nM siRNA, respectively. Cells were analysed at 48–72 hr post transfection.The Ambra1 binding site in FAK was identified using peptide arrays as published previously , 2007. BCells were washed twice in ice-cold PBS and then lysed in RIPA buffer  supplemented with PhosStop and Complete Ultra Protease Inhibitor tablets . Lysates were cleared by centrifugation at 10000 rpm for 15 min and analyzed by western blotting. Protein concentration was calculated using a BCA protein assay kit . For immunoprecipitation, 1 mg cell lysates were incubated with 2 μg of unconjugated or 10 μl of agarose-conjugated antibodies at 4°C overnight with agitation. Unconjugated antibody samples were incubated with 10 μl of Protein A or Protein G agarose for 1 hr at 4°C. Beads were washed three times in lysis buffer and once in 0.6 M LiCl, resuspended in 20 μl 2x sample buffer  and heated for 5 min at 95°C. Samples were then subjected to SDS-PAGE analysis using the Bio-Rad TGX pre-cast gel system. Proteins were immunoblotted using the Bio-Rad Trans-blot Turbo transfer system, blocked in 5% BSA in TBST (TBS supplemented with 1% Tween-20), and incubated with primary antibody overnight at 4°C. Blots were washed three times in TBST, incubated with peroxidase-conjugated secondary antibody for 45 min at room temperature, washed as before, developed using Clarity Western ECL Substrate  and imaged using a Bio-Rad ChemiDoc MP Imaging System .SCR_003032).For mass spectrometry, SCC FAK-WT and -/- cells were lysed in RIPA buffer. For the immunoprecipitations, 2 mg cell lysates (samples in triplicates) were incubated with 2 μg of unconjugated antibodies  or 10 μl of agarose-conjugated antibodies (FAK–agarose) at 4°C overnight with agitation. Unconjugated antibody samples were incubated with 20 μl of Protein A agarose for 1 hr at 4°C. Beads were washed twice in lysis buffer and twice in PBS. Protein complexes were subjected to on-bead proteolytic digestion, followed by desalting and liquid chromatography–tandem mass spectrometry as reported previously . The mas2 and 0.2% Triton X-100) for 10 min. Cells were subsequently washed twice in TBStx (TBS supplemented with 0.1% Triton X-100) and blocked in TBStx block (TBStx supplemented with 3% BSA). Primary antibodies were incubated in TBStx block overnight at 4°C, followed by three 5 min washes in TBStx, incubated with Alexa Fluor-labelled secondary antibodies diluted 1:200 in TBStx block  and washed as before prior to being mounted in Vectashield mounting medium containing DAPI . Cells were imaged using a FV1000 confocal microscope .Cells were grown on glass coverslips for 24 hr and washed once in TBS prior to fixation  microscopy an inverted IX81 microscope  with a 150 × 1.45 NA UAPON TIRF objective using 491 and 561 excitation lines was used. Colocalisation was analyzed using the ImageJ plugin JaCoP .SCR_003070).The intensity of pSrc or Paxillin at focal adhesions from three independent experiments  was measured using ImageJ  was added to the cells for 5 min. Cells were rinsed with PBS for 10 s using a Waterpik dental flosser set at 2, and the remaining attached focal adhesions were lysed in 2x sample buffer. Western blot bands were analysed by densitometry.For western blot analysis, focal adhesions were crosslinked with 3 mM dimethyl 3,3'-dithiobispropionimidate  for 30 min at room temperature and then quenched with 20 mM Tris-HCl, pH 8.0, for 5 min at room temperature. After another wash with 20 mM Tris-HCl, pH 8.0, cells were rinsed with ice-cold PBS, and 5 ml of focal adhesion extraction buffer  for 1 hr. Horizontal z sections through the Matrigel were acquired at 10 μm intervals with an Olympus FV1000 confocal microscope. The images were evaluated using ImageJ software.Invasion was analysed as described in . Briefly2O and dried at 37°C. The fixed cells were stained with sulforhodamine B (SRB) (0.4% SRB in 1% acetic acid) for 30 min, washed five times with 1% acetic acid and dried at 37°C. The SRB was dissolved in 10 mM Tris, pH 10.5, for 2 hr at room temperature and the absorbance was read at 540 nm. Samples were normalised to the 6 hr time point and relative adhesion was calculated by setting the FAK-WT values to 1.Cells were washed twice in PBS, resuspended in PBS and rotated for 45 min at 4°C. After that, 8000 cells/well were plated in serum-free medium on fibronectin-coated 96-well plates or on plastic at 37°C. Cells were fixed after 20 min, 1, 2 and 6 hr with 25% trichloracetic acid (TCA) and stored overnight at 4°C. Plates were washed five times with H2. After nine days, images were taken from 6–10 random fields (10x magnification) and colonies were counted.Cells were resuspended in a 1.4% methylcellulose solution in growth medium, plated on a layer of 0.9% agarose and incubated at 37°C, 5% COn = 5 (5 cells per experiment). For the invasion assay, the experiment was repeated with the following times: siAmbra1, n = 5; FAK P875A/P881A mutant, n = 8; siDctn1; n = 6 and siIFITM3, n = 6. All other experiments shown, n = 3. Quantification of internalised active Src was carried out by counting 100 cells and calculating the percentage. Error bars for the graphs showing the intensity of pSrc Y416 staining at focal adhesions and the invasion assays represent s.e.m. Error bars for all other experiments show s.d. Student’s t-test was carried out to calculate the statistical significance.For measuring the intensity of pSrc Y416 staining at focal adhesions (immunofluorescence),  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Tony Hunter (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This study examines Ambra1 in a new role as a Src/FAK complex protein. The novel concepts being addressed are very interesting and potentially of relevance to the control of both adhesion signalling and autophagy.Essential revisions:The reviewers agreed that the study is potentially interesting and represents a novel role for Ambra1, but some aspects of the data require clarification and strengthening to support the conclusions being proposed. The reviewers agreed that the following points should be addressed in a revised manuscript:1) The rationale for looking at Ambra1 is not explained, leaving the reader wondering why the authors landed upon Ambra1 as a modulator of FAK-dependent SRC activity? Did they perform a proteomic screen to look for FAK interacting proteins? This must be explained in more detail.2) Co-IPs throughout the manuscript lack quantification and IgG controls are missing from 3) In 4) 5) Co-localization of Ambra1 at focal adhesions in 6) Cells in 7) In several places, the authors use '% cells with internalised pSrc to make conclusions regarding active Src redistribution , 4C/F. T8) Ambra1 appears to co-IP with both LC3B-I and LC3B-II, which is not consistent with it forming a complex at mature autophagosomes in which case, one would only expect to detect LC3B-II co-IPing with Ambra1. The LC3B input lanes in 9) The Ambra-1 knockdown cells in 10) Src levels in Ambra1 knockdown cells should be quantified as one would expect differences if Src is no longer trafficked to the autophagosome.11) The significance of the FAK-/- cell findings would be greatly strengthened if Ambra1-dependent relocalization of Src to focal adhesions could be shown in detached cells.12) The rationale for selecting dynactin1 and IFITM3 for further study out of the many proteins identified by mass spec as complexing with Ambra1, pSrc and FAK should be explained.13) The potential for IFITM3 to localize to adhesions and the requirement for complex formation between pSrc and Ambra1 (within or outside adhesion complexes) should be tested? At present it is difficult to understand which proteins other than Ambra1 and FAK are directly interacting to mediate the specific effects.14) The authors do not show co-localization of endogenous Ambra1 and FAK or Src in whole cells or in cells expressing the P875A/P881A mutant FAK that would justify their model. These data should be provided.15) That Ambra1 is itself required for invasion, but a FAK mutant that does not bind Ambra1 promotes invasion is somewhat confusing. Both of these interventions result in increased pSrc at focal adhesions by disrupting the 'spatial rheostat'. The potential for Ambra1 compete with FAK for Src binding at adhesion and/or localize to adhesions in a FAK-independent manner should be discussed. Essential revisions:The reviewers agreed that the study is potentially interesting and represents a novel role for Ambra1, but some aspects of the data require clarification and strengthening to support the conclusions being proposed. The reviewers agreed that the following points should be addressed in a revised manuscript:1) The rationale for looking at Ambra1 is not explained, leaving the reader wondering why the authors landed upon Ambra1 as a modulator of FAK-dependent SRC activity? Did they perform a proteomic screen to look for FAK interacting proteins? This must be explained in more detail.We have explained this in more detail in the Results section . Essentially, we originally found Ambra1 in a phage display screen looking for novel FAK binding partners a few years ago, and this is now confirmed by reciprocal co‐IPs . As Ambr2) Co-IPs throughout the manuscript lack quantification and IgG controls are missing from We have added IgG controls for the FAK‐IPs in 3) In As Ambra1 shows dense diffuse intracellular staining, it was difficult to visualise any co‐localisation with FAK at focal adhesions in whole cells. Therefore, we isolated focal adhesions by hydrodynamic force. This has been re‐written in the manuscript . In addition though, we have now performed Total Internal Reflection Fluorescence (TIRF) microscopy , where w4) As many autophagosomal markers  have been reported at focal adhesions , we used a non‐autophagic, non‐adhesion cellular protein to demonstrate that the staining was not likely to be general cellular debris .5) Co-localization of Ambra1 at focal adhesions in The co‐localisation of Ambra1 at focal adhesions has been quantified and COSTES r values have been added.6) Cells in We do not see any consistent general differences in nuclear morphology or nuclear size. To be sure, we have now measured the size of the nuclei in FAK‐WT and ‐/‐ cells and could not detect any difference . HoweverThe size of nuclei from 50 SCC FAK-WT and -/- cells was measured using ImageJ. Error bars, s.d.7) In several places, the authors use '% cells with internalised pSrc to make conclusions regarding active Src redistribution , 4C/F. TThe co‐localisation of pSrc with Paxillin has been analysed, COSTES r values included and statistical tests performed see .8) Ambra1 appears to co-IP with both LC3B-I and LC3B-II, which is not consistent with it forming a complex at mature autophagosomes in which case, one would only expect to detect LC3B-II co-IPing with Ambra1. The LC3B input lanes in We have included a higher quality blot and densitometric quantification of the LC3B‐IP . The red9) The Ambra-1 knockdown cells in Paxillin staining has been included in the 10) Src levels in Ambra1 knockdown cells should be quantified as one would expect differences if Src is no longer trafficked to the autophagosome.Ambra1 was knocked down with a pool or two individual siRNAs and pSrc was measured relative to protein loaded (using GAPDH as an invariant protein) by immunoblotting . Quantif11) The significance of the FAK-/- cell findings would be greatly strengthened if Ambra1-dependent relocalization of Src to focal adhesions could be shown in detached cells.Unfortunately, when the cells are detached, they do not have discernable focal adhesions and so it has not been possible to check for this. We have stated this in the text .12) The rationale for selecting dynactin1 and IFITM3 for further study out of the many proteins identified by mass spec as complexing with Ambra1, pSrc and FAK should be explained.We selected these two trafficking proteins to address the hypothesis that Ambra1 was influencing the localization of active pSrc via binding partners involved in trafficking. Initially we selected IFITM3 because it was present in complex with all three proteins as seen by interaction proteomics . We init13) The potential for IFITM3 to localize to adhesions and the requirement for complex formation between pSrc and Ambra1 (within or outside adhesion complexes) should be tested? At present it is difficult to understand which proteins other than Ambra1 and FAK are directly interacting to mediate the specific effects.The IFITM3 antibody does not work well for immunofluorescence, which makes it difficult to show the localization of IFITM3 to focal adhesions. However, when we isolate focal adhesions by hydrodynamic force and analyse the lysates by immunoblotting, we detect IFITM3 in isolated focal adhesions . This imIFITM3 knockdown had no detectable impact on the Ambra1‐pSrc interaction , but kno14) The authors do not show co-localization of endogenous Ambra1 and FAK or Src in whole cells or in cells expressing the P875A/P881A mutant FAK that would justify their model. These data should be provided.We have now added data to show that Ambra1, Src and FAK co‐localise at focal adhesions by using TIRF microscopy, to overcome the problem of much diffuse and generally vesicular Ambra1 staining (now shown in 15) That Ambra1 is itself required for invasion, but a FAK mutant that does not bind Ambra1 promotes invasion is somewhat confusing. Both of these interventions result in increased pSrc at focal adhesions by disrupting the 'spatial rheostat'. The potential for Ambra1 compete with FAK for Src binding at adhesion and/or localize to adhesions in a FAK-independent manner should be discussed.Sorry, this was a bit confusing. To explain more clearly, Ambra1 is absolutely required for optimal invasion – since its depletion suppresses invasive migration. However, this is not linked to a greater amount of pSrc at focal adhesions in FAK‐WT cells as the reviewer suggests see . It is,


Diacylglycerol kinase (DGK) phosphorylates DG to generate phosphatidic acid. Recently, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the secondary spermatocytes and round spermatids of the testis. In this study, we cloned the full length DGKη3 gene and confirmed the endogenous expression of its protein product. During the cloning procedure, we found a new testis-specific alternative splicing product of the DGKη gene, DGKη4, which lacks half of the catalytic domain. We examined the DGK activity and subcellular localization of DGKη3 and η4. DGKη3 had almost the same activity as DGKη1, whereas the activity of DGKη4 was not detectable. In resting NEC8 cells (human testicular germ cell tumor cell line), DGKη1, η3 and η4 were broadly distributed in the cytoplasm. When osmotically shocked, DGKη1 and η4 were distributed in punctate vesicles in the cytoplasm. In contrast, DGKη3 was partly translocated to the plasma membrane and co-localized with the actin cytoskeleton. These results suggest that DGKη3 and η4 have properties different from those of DGKη1 and that they play roles in the testis in a different manner. Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to generate phosphatidic acid –6. DiacyWe recently reported that DGKη is expressed in stomach cancer and HeLa cervical cancer cells and that it is required for the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade, which is activated by epidermal growth factor . MoreoveSeveral reports have revealed that DGKη is abundantly expressed in the reproductive organs, testis and ovary –12. TherIn this study, we cloned the full length DGKη3 gene. Moreover, during the cloning procedure, we found a new testis-specific alternative splicing product of DGKη gene, DGKη4, which lacks the C-terminal half of DGKη3 and then examined the properties of DGKη3 and η4.2. cDNA was transfected into COS-7 cells by electroporation with a Gene Pulser XcellTM Electroporation System  according to the manufacturer's instructions.COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  at 37°C in an atmosphere of 5% CO2. The cells were transiently transfected with cDNA using FuGENE HD Transfection Reagent  according to the manufacturer’s instructions. Twenty-four hours after transfection, the cells were serum starved for 3 h and incubated in RPMI-1640 with 500 mM sorbitol for 30 min.NEC8  were obtained from Japanese Collection of Research Bioresources . The cells were maintained in RPMI-1640 medium  containing 10% fetal bovine serum at 37°C in an atmosphere of 5% COcDNAs encoding for mouse DGKη1 (1–1156 aa), DGKη3 (1–1125 aa) and DGKη4 (1–558 aa) were amplified by PCR using KOD-plus DNA polymerase . The products were inserted into the SalI/SacII site of pAcGFP-C1 vector .2) containing 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml each of leupeptin, pepstatin, aprotinin and soybean trypsin inhibitor and Complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics), and centrifuged at 1,000 x g for 5 min. The protein concentration in the supernatants was determined using a bicinchoninic acid protein assay kit . The tissue lysates (50 μg of protein) were separated on SDS-PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane . The membrane was blocked with 5% skim milk and incubated with an anti-DGKη polyclonal antibody ATP (100000 cpm/nmol). The reaction was initiated by adding the cell lysates, and it continued for 5 min at 30°C. Lipids were extracted from the mixture, and phosphatidic acid was separated by thin layer chromatography. The phosphatidic acid spot was scraped and counted by a liquid scintillation spectrophotometer.pAcGFP-DGKη1, η3, η4 or pAcGFP vector were transfected into COS-7 cells. After 48 h, the cells were harvested and suspended in ice-cold lysis buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM MgClStatistical comparisons were performed using one-way ANOVA followed by a Tukey’s test.We previously found a new alternative splicing product of DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues  Fig 1)..Fig 1). Although the DGKη3 mRNA was strongly expressed in the testis, its protein band has not been detected in our previous report . It is pWhen we performed RT-PCR for sequencing, we detected a longer DGKη mRNA (327 bp) that includes 35 bp of additional nucleotides derived from intron 14 . This inet al. reported that DGKη3 was not expressed in ovary or uterus whereas DGKη1 and η2 were expressed there. Thus, the expression of DGKη3 in brain, liver, kidney, lung, skeletal muscle, vesicula seminalis, prostate gland and epididymis was checked. We confirmed that the 735 bp band derived from DGKη3 mRNA was expressed in testis alone  and 735 bp (DGKη3) products, respectively, and were analyzed using primers C and D, which amplify 327 bp  and 292 bp (DGKη4) products, respectively. Shionoya is alone . On the The 327 bp product amplified from DGKη4 mRNA was also detected only in the testis . The 292We next characterized properties of the newly identified splice variants, DGKη3 and η4. To measure DGK activities of protein products encoded by DGKη3 and η4 genes, these proteins were overexpressed in COS-7 cells. We confirmed expression of DGKη3, η4 and η1, which is a positive control . CompareWe next tested whether DGKη3 and η4 respond to stress stimulation. To address this, we determined subcellular localization of DGKη3 and η4 in NEC8 cells (human testicular germ cell tumor cell line) in the presence and absence of 0.5 M sorbitol (osmotic stress). AcGFP alone were broadly distributed in the cytoplasm and nucleus in the presence and absence of 0.5 M sorbitol . In the In this study, we cloned cDNA of the testis specific full length DGKη3 mRNA Figs  and 2. MThe DGKη3 mRNA is a splice variant derived from the DGKη1 mRNA but not from the DGKη2 mRNA Figs . DGKη4 aet al. [The biochemical and cell biological properties of DGKη3 and η4 were different from those of DGKη1. First, DGK activity was not detectable for DGKη4 . The loset al.  revealedSecond, the subcellular localization of DGKη3 was different from that of DGKη1 . EnvironIt is possible that the splice variants DGKη3 and DGKη4 might be artifacts of cDNA preparation. However, this possibility is unlikely because the splice variants were detected only in the testis , althougDGKη3 and η4 are expressed in the testis , especia


Metabolic diseases are characterized by a decreased action of insulin. During the course of the disease, usual treatments frequently fail and patients are finally submitted to insulinotherapy. There is thus a need for innovative therapeutic strategies to improve insulin action. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter that specifically binds to the activated insulin receptor (IR) and inhibits its tyrosine kinase activity. Molecules disrupting Grb14-IR binding are therefore potential insulin-sensitizing agents. We used Structure-Based Virtual Ligand Screening to generate a list of 1000 molecules predicted to hinder Grb14-IR binding. Using an acellular bioluminescence resonance energy transfer (BRET) assay, we identified, out of these 1000 molecules, 3 compounds that inhibited Grb14-IR interaction. Their inhibitory effect on insulin-induced Grb14-IR interaction was confirmed in co-immunoprecipitation experiments. The more efficient molecule (C8) was further characterized. C8 increased downstream Ras-Raf and PI3-kinase insulin signaling, as shown by BRET experiments in living cells. Moreover, C8 regulated the expression of insulin target genes in mouse primary hepatocytes. These results indicate that C8, by reducing Grb14-IR interaction, increases insulin signalling. The use of C8 as a lead compound should allow for the development of new molecules of potential therapeutic interest for the treatment of diabetes. To overcome this defect, two different strategies are commonly used, aiming either at increasing insulin secretion or at improving distal insulin action, in order to decrease hepatic glucose production and enhance peripheral glucose utilization2. However, when the disease progresses, these treatments often become less efficient and type 2 diabetic patients need to resort to insulinotherapy. The identification of new molecular targets therefore remains an important issue for the development of innovative therapeutical approaches to improve insulin action3.Changes in lifestyle over the last decades resulted in a dramatic increase in the prevalence of metabolic diseases such as obesity and type 2 diabetes, which are characterized by an insulin-resistance state4. Binding of insulin to its receptor induces conformational changes that bring the two β-subunits nearer to each other, leading to trans-autophosphorylation of the β-subunits on tyrosine residues. The activated receptor then recruits and phosphorylates intracellular substrates such as IRSs and Shc, therefore initiating downstream signaling pathways  and promoting the physiological effects of insulin5. However, accurate regulation of insulin action results from a tight balance between activation of these signaling pathways and simultaneous induction of feedback mechanisms. These negative regulations involve the action of protein tyrosine phosphatases7 as well as adaptor proteins, like the Grb7 family of proteins9.The first step of insulin action is its binding to its membrane receptor (IR), a member of the tyrosine kinase family of receptors, and the activation of intracellular pathways leading to the final anabolic effects of the hormone10. Grb14 is selectively expressed in insulin-sensitive tissues and is rapidly recruited to the activated receptor upon insulin stimulation13. Grb14 binds mainly through its Between PH and SH2 (BPS) domain to the phosphorylated insulin receptor tyrosine kinase domain (IRTK) and acts as a pseudosubstrate to inhibit its catalytic activity15. Interestingly, recent genetic studies suggested an association between the locus of Grb14 and diabetes, insulin resistance or fat distribution18. Moreover, Grb14 expression is increased in adipose tissue of insulin-resistant animal models and type 2 diabetic patients, and improvement of insulin sensitivity in ob/ob mice by treatment with an antidiabetic drug (thiazolidinedione) is associated with a reduction in Grb14 expression19. Grb14 expression is also increased in skeletal muscle of obese and insulin-resistant patients and decreased by weight loss, along with increased insulin sensitivity20. All these data suggest that this protein could be involved in the decrease in insulin signaling observed in metabolic diseases. Targeting IR-Grb14 interaction, in order to relieve the Grb14 inhibitory action on the IR, may thus represent a new strategy to improve insulin signaling and action.Adapters of the Grb7 family, which includes Grb7, Grb14, and Grb10, are involved in the regulation of tyrosine kinase receptor signaling24. After a structural analysis of docked molecules present in the ChemBridge database in two different pockets at the surface of the IR, we selected a list of 1000 molecules that were experimentally tested for disrupting the Grb14-IR interaction in an a-cellular BRET (Bioluminescence resonance Energy Transfer) assay26. Three compounds were isolated and further characterized for their activity on insulin signaling.To identify small non-peptidic inhibitors of the IR-Grb14 interaction, we performed structure-based virtual ligand screening experiments, based on the Grb14(BPS)-IR crystallographic structure15. Two binding pockets were identified and used for screening  and 60%. Of note, C8 also induced a significant decrease (50%) in the BRET signal in the absence of insulin stimulation. In co-immunoprecipitation experiments, as expected, the association between IR and Grb14 was also much more prominent with purified receptors from insulin-stimulated HEK-293T cells compared to purified receptors from non-stimulated cells. C1 and C8 reduced respectively by 30% and 60% the amount of IR co-precipitated with Grb14, while C5 had no effect. C8 was therefore the most efficient compound to inhibit insulin-induced IR-Grb14 interaction, both in BRET and co-IP experiments.We have scored a collection containing 340 000 compounds (ChemBridge) after structured-based virtual screening performed on the basis of the Grb14(BPS)-IR crystallographic structureles Fig. , expecteles Fig.  and co-iles Fig.  experimeIn an attempt to identify compounds with improved inhibitory properties, similarity search was performed on the basis of C1, C5 and C8 structures. The ChemBridge database  was scanned for compounds with shape similar to C1, C5, or C8, or compounds similar to both C1 and C5. Using structured-based and ligand-based virtual screening and different virtual filters, 51 molecules with high-score for potential inhibition of the IR-Grb14 interaction were selected, purchased and experimentally evaluated for inhibition of IR-Grb14 interaction using the a-cellular BRET assay Fig. . This grin silico analysis of the molecules C1 and C5 (and molecules similar to these two compounds) suggested that under some experimental conditions these molecules could interfere with some assays28. In this context, we decided thereafter to focus on the investigation of the inhibitory activity of C8.Thus, from our initial screen and the following search for compounds with structural similarity with the 3 initial hits, we identified 5 compounds effective in decreasing the IR-Grb14 interaction. C8 remained the most active, as we did not succeed to find a more active compound among C1, C5 and C8-derived compounds. Furthermore, In the initial screen the compounds were incubated with IR before adding Grb14, implying that they acted by preventing the association between the two proteins (Protocol 1). To check this model, two other BRET protocols were tested in the same experiment Fig. . In “ProE)-3-(5-methyl-2-furanyl)-2-propenoic acid (named “compound B”). The second step consisted in the condensation of the antibiotic with the acid in the presence of a peptide coupling reagent  and 5-methyl-2-furfuraldehyde in two steps. The first step was the conversion of 5-methyl-2-furfuraldehyde into , immunoprecipitations were performed in cells co-expressing the IRTK-Rluc and the Grb14-YFP chimeric proteins and which were treated or not with C8 , in the absence or the presence of C8 (50 or 100 µM). A 7-fold increase in the cell population was observed when cells were cultured with 0.1% serum and a 10-fold increase was observed with 1% or 10% serum. The addition of C8 did not alter cell growth both at low- and high-serum concentration . C8 had no effect on basal BRET (17.3 ± 2.4 mBU). As shown previously, binding of insulin to its receptor stimulated BRET signal. The addition of C8 did not alter this insulin-induced BRET signal, suggesting that the conformational changes induced by insulin binding were not modified in the presence of C8  production  to the plasma membrane in response to PI-3 kinase-induced phosphatidylinositol-3,4,5 triphosphate , which binds to activated IR and dephosphorylates the tyrosine residues present in the activation loop34. Oscillation of the IR between inactive and active forms was clearly demonstrated by experiments showing that treatment of cells with vanadate, a protein tyrosine-phosphatase inhibitor, led to a significant increase in IR phosphorylation and in its tyrosine kinase activity, even in the absence of insulin36. Thus, in the absence of hormonal stimulation the IR undergoes cycles of phosphorylation/dephosphorylation, allowing Grb14 to bind when it is in its active conformation. The compound C8 could therefore inhibit a basal interaction between the IR and Grb14, explaining its activity observed in the absence of insulin using various experimental systems  or high glucose plus insulin (G25i) concentrations, mimicking respectively the fasted and fed states, with or without C8. We first investigated the effect of C8 on insulin-induced activation of Akt, the major player in the transmission of insulin metabolic effectsion Fig. . The sti60% Fig. . To stud60% Fig. . Altoget3 by PI3-kinase. Importantly, studies on primary hepatocytes showed that C8 increased the effect of insulin on the expression of its target genes. It should be underlined that an increased lipogenesis induced by C8 may lead to the accumulation of triglycerides in liver cells and cause hepatic steatosis, which could be a problem in the context of diabetes therapy. However, hepatic steatosis is not necessarily associated with the development of insulin resistance, as illustrated by an increasing number of studies38. More particularly, increased levels of monounsaturated fatty acids newly synthesized through the de novo lipogenic pathway can exert a protective effect on liver insulin sensitivity39. It would thus be interesting to analyze the effects of C8 on the potential development of hepatic steatosis and on the composition of liver lipid stores in mice.Considering the epidemic expansion of obesity and type 2 diabetes in this beginning century, two pathologies associated with an insulin-resistance state, the identification of new molecules capable of improving insulin action is of considerable medical interest. In the present study, using a structure-based virtual screening to search for molecules blocking Grb14 binding to the receptor and improving insulin signaling we identified a chemical probe, C8, that reduced IR-Grb14 interaction, stimulated the MAP kinase pathway and induced the production of PIP44. Inhibition of IR-Grb14 may be an additional example of the potential of this strategy for the development of small molecules of therapeutic interest for the treatment of important human pathologies.Protein-protein interactions play a central role in a variety of biological processes and structures. The inhibition of protein-protein interaction by small molecules has long been thought to be difficult to achieve, mostly due to the large and relatively flat and featureless interfacial areas involved. However, the development of protein-protein interaction inhibitors offers attractive opportunities for novel therapeutic intervention and some successes have recently emerged in the field of inflammation , apoptosis  or virology (HIV-1 protease)All methods were carried out in accordance with relevant guidelines and regulations.15  was used for in silico screening. A multi-step docking/scoring protocol employing MS-DOCK45 and Surflex46 was used. Two binding pockets could be identified at the surface of the IR and the compound collection was docked on these two different pockets. The ChemBridge database (the starting collection contained about 500 000 unique molecules) was filtered with our software FAF-Drugs47 and an in-house “soft” drug-like physicochemical property filter (http://fafdrugs4.mti.univ-paris-diderot.fr/filters.html) resulting in a final collection of 340,000 molecules. The 3D generation was performed with our in-house program DG-AMMOS48.The crystallographic structure of the activated IRTK in complex with the Grb14 BPS domainE)-3-(5-methyl-2-furanyl)-2-propenoic acid (compound A) was prepared from 5-methyl-2-furfuraldehyde according to a published procedure49.Sulfafurazole (compound B) and all other chemical reagents and solvents were obtained commercially (Sigma-Aldrich).   followed by N,N-diisopropylethylamine . The mixture was stirred for 5 min at 0 °C, then sulfafurazole  was added. The mixture was stirred at room temperature for 15 min and then irradiated  for 25 min in a microwave reactor (CEM Discover). The mixture was digested with ethyl acetate (100 ml) and extracted with a saturated aqueous NaHCO3 solution (50 ml). The aqueous extract was treated with 6 M HCl (6 ml) so that acidic pH was obtained and a precipitate appeared. The precipitate was isolated by Buchner filtration, rinsed with distilled water (2 × 5 ml) and dried at 40 °C for 6 h to provide C8  as a yellow solid; mp = 150–151 °C; Rf = 0.33 (EtOAc); IR (ATR) ν: 3315, 3112, 3060, 2839, 2774, 1673, 1625, 1589, 1540, 1526, 1497, 1419, 1368, 1334, 1348, 1253, 1189, 1162 cm−1; 1H NMR  δ: 10.95 , 10.60 , 7.88 , 7.72 , 7.37 , 6.80 , 6.37 , 6.28 , 2.35 , 2.10 , 1.63 ; 13C NMR  δ: 164.2, 161.4, 155.6, 154.9, 149.4, 143.7, 133.5, 128.2, 128.0, 118.8, 117.1, 116.9, 109.2, 105.1, 13.5, 10.3, 5.8; HRMS: m/z found: 424.0925 [M + Na]+; C19H19N3NaO5S requires 424.0938.In a 10 ml glass vessel, a solution of compound A  in dry dimethylformamide (625 μl) under argon at 0 °C was treated with 1-bis(dimethylamino)methylene-1HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium containing 4.5 g/l glucose and 10% fetal bovine serum (FBS). Cells were transfected using FuGENE 6 (Promega) according to the manufacturer’s instructions. αML12 cells were cultured in DMEM/Ham’s F12 medium containing 10% FBS and supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 µg/ml selenium, 40 ng/ml dexamethasone, 100 units/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in serum free medium and stimulated with or without 5 nM insulin and 50 µM of compound C8 for 4 h at 37 °C. Cells were incubated in serum free medium and stimulated with or without 100 nM insulin and 25 µM of compound C1-13 for 4 h at 37 °C.50. Cells were cultured in Medium 199. For lipogenic gene expression analysis, cells were cultured for 24 h in the presence of 5 mM glucose or 25 mM glucose/10 nM insulin in the absence or the presence of 50 µM of compound C8. For gluconeogenic gene expression analysis, cells were preincubated for 1 h in the presence of 5 mM glucose with or without 10 nM glucagon and then treated for 8 h in the absence or the presence of 1 nM insulin and 50 µM of compound C8.Mouse primary hepatocytes were isolated as described previously51. The cDNA coding for Raf-RBD-Luc was obtained by fusing the Ras Binding Domain (RBD) of Raf to the N-terminus of Renilla luciferase. YFP-Ras plasmid was provided by Van der Meer W.R., University of Leiden. YFP-Grb10 plasmid was provided by Liu F., University of Texas. The cDNA coding for IRTK48-Rluc was obtained by fusing the tyrosine kinase domain of the insulin receptor to the N-terminus of Renilla luciferase.IR, IR-Rluc, Grb14-YFP, YFP-PTP1B, YFP-targeted to the plasma membrane and Luc-Akt-PH expression vectors have been described previously32. After elution with N-acetylglucosamine (0.5 M), fractions enriched in Rluc activity were pooled, aliquoted and stored at −80 °C for subsequent use.HEK-293T cells were transfected with 6 μg of IR-Rluc cDNA, 6 μg of Grb14-YFP or 6 μg of empty vector cDNAs per 100 mm dish. Two days after transfection, cells transfected with Grb14-YFP or empty vector were lysed, cleared and extracts were aliquoted and stored at −80 °C for subsequent use. Cells transfected with IR-RLuc were stimulated or not with 100 nM of insulin during 10 minutes before lysis. Fusion receptors were partially purified by chromatography on wheat-germ lectin Sepharose (WGL) as described previously52.A-cellular BRET assay was performed using 20 µl of IR-Rluc eluate preincubated in 96-well microplates for 20 min at room temperature with different concentrations of the compounds to be tested, or DMSO as control. 20 µl of extract obtained from Grb14-YFP transfected cells or control extract (from empty vector transfected cells) were added, and after 40 min of incubation, the substrate of luciferase cœlenterazine  was added . Light emission acquisition was then performed at 480 and 530 nm. The BRET signal was expressed in milli-BRET units, corresponding to the BRET ratio multiplied by 1000. The BRET ratio was previously defined as the emission ratio 530/480 nm obtained when the two partners are coexpressed, corrected by the ratio 530/480 nm obtained for the luciferase-fused protein expressed aloneIn vitro measurement of BRET signal was performed using 5–10 μl of WGL eluate (approximately 2 μg of proteins) preincubated in 96-well microplates for 30 min at room temperature in a total volume of 55 μl containing 30 mM MOPS, 1 mM Na3VO4, and different concentrations of ligands. After preincubation, coelenterazine was added . Light emission acquisition (at 480 and 530 nm) was then started.HEK-293T cells were transfected with 2.5 μg of IR-Rluc cDNA or with 1 μg of IR-Rluc and 1.5 μg of IR-EYFP cDNAs per 12-well plate. 48 h after transfection, cells were extracted in buffer containing 1% (w/v) Triton X-100, 20 mM MOPS, 2.5 mM benzamidine, 1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and 1 μg/ml each aprotinin, pepstatin, antipain, and leupeptin. Fusion receptors were partially purified by chromatography on WGL as described above. 5 cells per 12 well plate and transfected 24 h later using FuGene 6 and the following combinations of vectors: i) Raf-RBD-Luc (400 ng), IR (400 ng) and either empty vector or WT or mutated pEYFP-RasN17 (400 ng); ii) Luc-Akt-PH (700 ng) and either empty vector or YFP-mem . BRET measurements on intact cells were performed essentially as described previously33. Briefly, the day after transfection, cells were detached with Trypsin-EDTA (Invitrogen) and resuspended in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g/l glucose and 10% fetal bovine serum. Cells were then transferred into 96-well microplates at a density of 30,000 cells per well. Twenty four hours later, cells were incubated in PBS at room temperature for 10 min with cœlenterazine at a final concentration of 2.5 μM. Cells were treated in the absence or the presence of different concentrations of ligands (insulin and test compounds), and light-emission acquisition was then started. The interaction was monitored for at least 30 min using the Mithras LB 940. The BRET signals were expressed as described above.HEK-293T cells were seeded at a density of 1.5 × 1053. Cell lysates were analyzed using 8% SDS–PAGE gels and immunodetected with the following antibodies: anti-GFP (Roche Diagnostics), anti-Luc (Chemicon), anti-IR , anti-4G10 (Santa Cruz Biotechnology), anti-3PY-IR (Biosource), anti-pAkt , anti-Akt1/2/3 .For protein analysis, cells were lysed on ice for 15 min in 200 µl lysis buffer/well containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Triton-X-100, 50 mM NaF, 10 mM β-Glycerophosphate, 1 mM Orthovanadate and a protease inhibitor cocktail  and extracts were cleared by a 15,000 × g centrifugation at 4 °C for 15 minFor co-immunoprecipitation assays of IR with Grb14, Wheat Germ Lectin (WGL)–partially purified receptors prepared from control or insulin-stimulated cells were preincubated for 20 minutes with control DMSO or with different concentrations of compounds, and the Grb14-YFP protein extract was added for 40 min. Protein extracts were then incubated for 1 h at room temperature with 5 μl anti-GFP antibody in the presence of protein G-agarose beads. The beads were washed three times in lysis buffer and the resulting immunoprecipitates were submitted to SDS-PAGE analysis and immunodetected with the indicated antibodies. Quantifications of Western blots represent the signal corresponding to immunoprecipitated Grb14 adjusted to the signal corresponding to immunoprecipitated IRβ.For co-immunoprecipitation assays of IRTK with Grb14 or Grb10, HEK-293T cells were transfected in 6-well plates using FuGENE 6 with 600 ng of IRTK48-Rluc plasmid and 600 ng of Grb14-YFP or YFP-Grb10 plasmid. Two days after transfection, cells were stimulated with 50 µM of compound C8 for 4 h. Cells were lysed as described above and protein and GFP contents of the lysates were quantified respectively using the BCA Protein Quantification Kit and the Fusion microplate analyzer. About 500 μg of proteins were incubated for 2h30 min at 4 °C with 5 μl of anti-GFP antibody in the presence of protein G-agarose beads. The beads were washed and bound proteins were analyzed as described above.32. Autophosphorylation of the receptors was assessed by immunoblotting using the 4G10 antiphosphotyrosine antibody.Partially purified IR-Rluc fusions (5 μl) were preincubated for 30 min at room temperature in a total volume of 60 μl containing 20 mM Tris (pH 7.4), 12 mM MgCl2, 2 mM MnCl2, and different concentrations of ligands (5 or 10 nM insulin and 50 µM of compound C8). The autophosphorylation reaction was initiated by adding 5 μl of ATP  for 10 min and stopped by the addition of SDS-polyacrylamide gel electrophoresis sample buffer54. After 3 washes, proteins were eluted from the beads in Laemmli buffer. Autophosphorylation of the endogenous receptors was assessed by immunoblotting using the anti-3PY-IR antibody.For autophosphorylation of endogenous insulin receptors in intact cells, HEK-293T cells were incubated for 5 min in the absence or the presence of 5 nM insulin and 50 µM of compound C8 and then lysed as described above. Clarified extracts (500 µg of proteins) were immunoprecipitated with anti-IR antibody and protein G-sepharose beads for 2 h 30 min at 4 °C55. Quantitative PCR was performed using a Lightcycler system and SYBR Green detection of amplified products (Roche Applied Science) using the following primer sequences, and gene expression was normalized over 18S RNA levels: steroid responsive element binding protein-1c (SREBP-1c), forward 5′-GACCCTTCCAGGAAACACTC-3′, reverse 5′-TGTTTGTTCTAGGGGCTGC-3′; acetyl CoA carboxylase (ACC), forward 5′-GAGGTGGCTAAGAGGAGGCTCT-3′; fatty acid synthase (FAS), forward 5′- TTCCAAGACGAAAATGATGC-3′, reverse 5′-AATTGTGGGATCAGGAGAGC-3′; steroyl CoA desaturase-1 (SCD1), forward 5′-CCGGAGACCCTTAGATCGA-3′, reverse 5′- TAGCCTGTAAAAGATTTCTGCAAA-3′; phosphoenolpyruvate carboxykinase (PEPCK), forward 5′-TGGCTACGTCCCTAAGGAA-3′, reverse 5′-GGTCCTCCAGATACTTGTCGA-3′; glucose-6-phosphatase (G6Pase), forward 5′-TTACCAGCCTCCTGTCGG-3′, reverse 5′- GACACAACTGAAGCCGGTTAG-3′; 18S, forward 5′- CCATCCAATCGGTAGTAGCG-3′, reverse 5′- GTAACCCGTTGAACCCCATT-3′.Total cellular RNA was extracted from cultured hepatocytes using the TRIZOL reagent and reverse-transcribed as described previously4 cells/well). Three replicate wells were used for each experimental condition.Cell viability was assessed after 72 h of culture using the Uptiblue assay (Interchim) according to the manufacturer’s instructions. Cells were seeded in 96-well plates . Blots from Fig. TM FLA 9000 .Blots from Figs t test or a one-way analysis of variance (ANOVA) followed by appropriated post-test as indicated in figure legends.Statistical analyses were performed with Prism software (GraphPad) using either a Supplementary dataset 1


However, the humanin-mediated signaling pathways are not well understood. In this paper, we demonstrate that humanin acts through the GP130/IL6ST receptor complex to activate AKT, ERK1/2, and STAT3 signaling pathways. Humanin treatment increases phosphorylation in AKT, ERK 1/2, and STAT3 where PI3K, MEK, and JAK are involved in the activation of those three signaling pathways, respectively. Furthermore, old mice, but not young mice, injected with humanin showed an increase in phosphorylation in AKT and ERK1/2 in the hippocampus. These findings uncover a key signaling pathway of humanin that is important for humanin's function and also demonstrates an age-specific in vivo effect in a region of the brain that is critical for memory formation in an age-dependent manner.Humanin is a small secreted peptide that is encoded in the mitochondrial genome. Humanin and its analogues have a protective role in multiple age-related diseases including type 2 diabetes and Alzheimer's disease, through cytoprotective and neuroprotective effects both  Humaninectively , 6. Humaectively , 8. The ectively  and theiectively . The lonectively –13. Humaectively , 14–16. response . Becausein vitro humanin treatment increases AKT-1 phosphorylation in mouse primary cortical neurons, and in vivo humanin injection also elevates AKT-1 phosphorylation after cerebral I/R injury while decreasing infarct volume [et al. showed that humanin binds to the formyl-peptide receptor-like receptor 1 (FPRL-1) to inhibit A-β induced cell death [2+ flux and activation of extracellular signal-regulated kinase (ERK 1/2) [et al. showed that knockdown of the mouse counterpart of FPRL-1, FPR2, did not attenuate humanin's neuroprotective effect against AD-related insults, suggesting that there was another receptor for humanin other than FPR2 [In terms of signaling, t volume . In moust volume . Ying etll death . Upon acERK 1/2) . Howeverhan FPR2 . Their ghan FPR2 .GP130 is a transmembrane protein and serves as the signal transduction unit of the IL-6 receptor family . IL-6 bivia CRE-mediated transcription in the hippocampus [Extracellular signal-regulated kinase (ERK1/2), a member of the mitogen-activated protein kinase pathway, is involved in many fundamental cellular processes including cell proliferation, survival, differentiation, mobility, and apoptosis , 28. An pocampus , 30. Anopocampus .in vitro and in vivo in multiple models.Humanin protects against cellular stress and improves pathologies in multiple age-related diseases including AD and diabetes, and we have previously shown that humanin activates intracellular signaling in pancreatic beta cells . Neverth® Pathway Analysis (IPA) to attempt to identify the humanin signaling pathway. The HNG targets can be categorized into several different molecular functional types: kinase, transcription regulator, transmembrane receptor, and so on  treatment for 30min in serum free conditions by using the Phospho Explorer Antibody Array. HNG is generated by replacement of Ser14 with glycine, and this substitution increases humanin activity [n Figure . The HNGn Figure . The IPAn Figure .473 and Thr308, MAPK p44/42 (ERK 1/2) at Thr202/Tyr204, and STAT3 at Tyr705 and Ser727. We examined MAPK p44/42 as it is a common downstream pathway of both IL-6 and PI3K/AKT as shown in the IPA canonical pathway analysis. HNG treatment caused a rapid increase in phosphorylation of ERK 1/2 at its regulatory Thr202/Tyr204 site in both serum free and complete media condition . Dimerization and DNA binding of STAT3 require phosphorylation of its Tyr705 site and mitochondrial localization of STAT3 requires phosphorylation of its Ser727 site [IPA canonical pathway analysis suggested that the IL-6, JAK/STAT3, and PI3K/AKT pathways were activated in response to treatment with HNG. ERK, AKT, and STAT3 were activated as a common component in those signaling pathways. To validate the predicted humanin signaling pathway in SH-SY5Y cells, we treated them with 100μM HNG in serum free or complete media for the indicated time periods. We then analyzed the phosphorylation of AKT at Sern Figure . Becausen Figure . HNG tre Figures . HNG sige Figure , whereas727 site , 34. Thi202/Tyr204), AKT (Ser473), and STAT3 (Tyr705). HNG treatment showed a rapid increase in phosphorylation of ERK 1/2 at its regulatory Thr202/Tyr204 site in both serum free and complete media conditions  has not yet been fully determined, we examined whether the canonical pathways regulate humanin-mediated AKT, ERK 1/2, and STAT3 activation. To accomplish this, we used specific inhibitors, LY294002, PD98059, and JAK I, to block PI3K, MEK, and JAK signaling, respectively. SH-SY5Y cells were pretreated with 10 μM LY294002, 10 μM PD98059, or 1μM JAK I for 30 min, followed by HNG treatment in serum free condition. LY294002 significantly inhibited phosphorylation of AKT, but not ERK Figure . PD98059JAK is constitutively associated with GP130, and humanin is known to bind to the trimeric receptor, which is comprised of IL-6 receptor family subunits including GP130, CNTFR alpha, and WSX-1 , 36. In The subcellular localization of ERK plays a crucial role in its signaling function, particularly in achieving its specificity . ERK is et al. showed that humanin injection into 3xTg AD mice improves spatial learning and memory deficits, while reducing Aβ plaque accumulation and the neuro-inflammatory response [The ERK signaling pathway regulates a variety of cellular processes including proliferation, differentiation, and cellular metabolism. In the hippocampus, ERK activation is involved in synaptogenesis and memory function. Niikura response . To examin vivo studies, demonstrate that humanin can specifically stimulate AKT and ERK 1/2 in the hippocampi of older mice, restoring the levels back to a youthful state, counteracting the decline in AKT phosphorylation with age [Humanin levels decline with age in tissue and plasma of both, rodents and humans . Mitochowith age . We findNotably, we find that the levels of phosphorylated AKT and ERK 1/2 increase in the hippocampi of older mice upon HNG treatment. These results suggest that humanin could improve cognitive function during aging and age-related diseases like Alzheimer's disease through these signaling pathways . Why humversus transient activation. It is also possible that the different ligands that engage GP130 have different effects on cellular signaling. Alternatively, humanin's in vivo effects on IGF-I signaling could be the primary mediator of these beneficial effects. Further studies will be needed to examine this in more detail and dissect apart the possible therapeutic uses of humanin.Our study implicates GP130 signaling as a key player in the beneficial effects of humanin action, and identifies humanin as a major GP130 agonist. In fact, transient activation of GP130 has many overlapping effects with humanin administration such as protection from doxorubicin toxicity, protection from ischemia/reperfusion injury, and metabolic effects including increasing insulin sensitivity –51. For 202/Tyr204) antibody (Cat. #4370S), anti-STAT3 antibody (Cat. #8204), anti-phospho-STAT3 (Tyr705) antibody (Cat. # 8204), anti-phospho-STAT3 (S727) antibody (Cat. #9134S), anti-GAPDH antibody (Cat. #5174S), and anti-Lamin B1 antibody (Cat. #12586). These antibodies are manufactured by Cell Signaling Technology . Rabbit anti-GP130 antibody (Cat. #SC-655) and mouse anti-Tom20 antibody (Cat. #SC-17764)  were also used.HNG  and humanin were synthesized and received from Genscript  and New-England Peptide . HNG was dissolved in Milli-Q water. PD98059, LY294002, JAK inhibitor I , and SC-144 hydrochloride  were used for the inhibitor experiments. The following antibodies were used in this study: anti-AKT (pan) antibody (Cat. #4691S), anti-phospho-AKT (Ser473) antibody (Cat. #4060S), anti-phospho-AKT (Thr308) antibody (Cat. #13038P), anti-p44/42 MAPK (Erk1/2) antibody (Cat. #9102S), anti-phospho-p44/42 MAPK (Erk1/2) supplemented with 10% fetal bovine serum  at 37°C in 5% COAfter peptide treatment for the indicated time periods, cytosolic and nuclear fractions of HEK293 and SH-SY5Y cells were obtained as previously described with some modifications . Briefly+ imager. If necessary, relative intensities of band in each condition were measured using Image J, a free software program provided by National Institute of Health .Cells were lysed with RIPA buffer  containing 0.1% SDS plus the Halt protease & phosphatase inhibitor cocktail. The lysates were homogenized using a sonicator, and the supernatant was collected by centrifugation at 15,000 x g for 15min at 4°C. For tissue, dissected hippocampi were lysed with RIPA buffer and homogenized using a tissue homogenizer followed by sonication. The supernatant was collected by centrifugation at 15,000 x g for 15min at 4°C. Protein content in the cellular lysates was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). Predetermined amounts of proteins (10-30μg) were separated on 4-20% SDS-PAGE gels and blotted onto PVDF membranes . Membranes were incubated with primary antibody at 4°C overnight according to the manufacturer's instructions. After several washes with Tris-buffered saline containing 0.1% Tween-20, membranes were incubated at room temperature (RT) for 1hr with the appropriate HRP-conjugated secondary antibody. Enhanced chemiluminescence was used for detecting specific bands. Membranes were imaged on a Bio-Rad ChemiDoc XRS+ imager.A PathScan RTK Signaling Antibody Array kit  was used according to the manufacturer's instructions. Briefly, cells were washed with ice-cold PBS and lysed in 1X Cell Lysis Buffer to collect cell lysates of control and HNG treated sample for the indicated time. The supernatant was collected by centrifugation at 15,000 x g for 15min at 4°C. Protein content in the cellular lysates was quantified using a Pierce™ BCA Protein Assay Kit. The Array Blocking Buffer was added to the array wells for 15min and predetermined amounts of protein (50μg) were incubated at 4°C overnight. After washing the array wells, the Detection Antibody Cocktail was added to the wells and incubated for 1h at RT followed by incubation with horseradish peroxidase-linked streptavidin for 30min at RT. The plates were then covered with the LumiGLO/peroxide reagent and imaged in the Bio-Rad ChemiDoc XRSn = 16) and 18 months (n = 10) of age were obtained from Jackson Laboratory  and the National Institute of Aging (NIA), respectively. The mice were singly housed under standard 12-hr light-dark cycle with access to water and rodent food ad libitum . Mice were randomly assigned to one of two experimental groups: a control group receiving daily Intraperitoneal (IP) injection of vehicle (sterilized water); a HNG-treated group receiving daily IP injection of 5mg HNG per kg body weight. Mice were euthanized after two weeks of treatment. Prior to euthanasia, these mice were fasted for 9 hours and were injected with HNG 15-30 minutes just before anesthesia with isoflurane. Tissues were collected, flash-frozen in dry ice and stored at −80°C.Male C57BL/6 mice at 12 weeks . A spreadsheet containing the list of humanin-mediated phosphorylated proteins was generated by using Partek® Genomomics Suite  and was uploaded into IPA. The criteria for the selection of the proteins of interest were an absolute fold change > 1.2 between control and HNG treated groups and P < 0.05. The software mapped each of the proteins to the repository of information in the Ingenuity Pathways Knowledge base. Molecular networks and canonical pathways regulated by humanin were obtained using IPA core analysis. Category rankings were based on the p values derived from the Fisher's exact test and the cut-off threshold for significance was a P-value < .05. Ratios were calculated as follows: number of genes in a given pathway that meet the cutoff criteria, divided by the total number of genes that make up that pathway and that are in the reference gene set.The specific humanin-mediated signaling pathways identified using the Phospho Explorer Antibody Array  were analyzed using Ingenuityt-tests, one-way ANOVA followed by Tukey's post hoc test, and two-way ANOVA followed by Bonferroni post-tests using GraphPad Prism 5 software. Values of * < 0.05, ** < 0.01, *** < 0.001 were considered statistically significant.Data are presented as mean ± S.E.M. Significant differences were determined by Student's


A significant literature has employed fragment models and a single adsorbed H2 to identify adsorption sites within IRMOFs, as well as the necessary adsorbate–adsorbent interactions needed to reach sufficient adsorption enthalpy for practical usage, however at high pressures it remains to be seen if H2···H2 intermolecular interactions may influence the energetics. This study focuses upon IRMOF-1 , and examines the individual H2 stabilization energies at different sites using Möller–Plesset perturbation theory and density functional theory alongside chemical models that consist of isolated fragment models and a cubic super cell cluster consisting of both the face- and edge-cube’s of IRMOF-1. Optimization of twenty stable configurations of singly adsorbed H2 in the super-cell cluster is observed to be essential to obtain energy ordering of the five primary sites consistent with experiment and prior benchmark calculations (α >> β > γ > δ ≈ ε). To examine site-to-site interactions that may occur in the high-pressure regime, 64 co-adsorbed H2 within a super-cell cluster have been studied . There, delocalization and/or charge transfer of electrons is observed from the σ orbitals of the H2 bound at the γ positions into the σ* orbitals of H2 bound at the α sites leads to stabilization of the interaction of H2 at the γ, by 1.4 kJ/mol, respectively (using M06-2X/LANL2DZ). This effect has been confirmed to be charge transfer, and not a manifestation of enhanced dispersion at high loading, through natural bond order (NBO) analysis and by comparisons of the square of off-diagonal NBO Fock matrix elements for both density functionals that account for dispersion interactions and Hartree–Fock calculations that ignore dispersion.Isoreticular metal organic frameworks (IRMOFs) have shown high uptake capabilities for storage of H Th. Th30]. The difference of the SE values for each site before and after simultaneous optimization is used to represent the change of the stabilization energy as a result of maximal loading. The interaction between hydrogen molecules was further investigated by NBO analysis . All the2 configurations [2 configurations at α, β, γ and δ sites using frag1 and frag2 with MP2/def2-TZVP, and the SE values calculated with MP2/aug-cc-pVTZ  of parallel (||) and perpendicular (⏊) Hurations ,13,14, Surations  has opti-cc-pVTZ . These d7 kJ/mol ,34 and tc linker . As suchnd frag2 , howeversis sets  using M0Both M06-2X and ωB97XD with LANL2DZ basis set represent significant improvements to the PBE data, being very close to the MP2 results, and having average errors relative to MP2 of 1.05 kJ/mol and 1.83 kJ/mol, respectively for ωB97XD and M06-2X functionals. The SE values using M06-2X/LANL2DZ are 11.13 kJ/mol, 3.77 kJ/mol, 3.47 kJ/mol, and 3.17 kJ/mol at the α, β, γ and δ sites, respectively. This represents the same order of stability as experiment (α >> β > γ > δ ≈ ε) . Increas2 on benzene also indicate that the perpendicular configuration is the favored geometry [The largest impact observed when using the cc-pVDZ-PP basis set comes from the relative energy of the α site—leading to the prediction that the most stable configuration is parallel rather than perpendicular. The other SE values on the β, γ, δ and ε sites do exhibit small changes when the basis set is increased from LANL2DZ to cc-pVDZ-PP. While they are of a small magnitude, they do change the order of energetic stability. Specifically, ωB97XD/LANL2DZ predicts that the δ(||) to be more stable than the δ(⏊), however increasing the basis to cc-pVDZ-PP reverses this trend. High level MP2 calculations of the parallel and perpendicular adsorbed configurations of Hgeometry ,18,35. C2 configurations and predict their interaction strengths at an improved computational cost relative to ωB97XD, however it has slightly poorer performance.Compared to the MP2 results, both M06-2X and ωB97XD using the cc-pVDZ-PP basis set exhibit somewhat improved performance relative to the prior PBE + dispersion calculations. These data further support the fact that it is important to use DFT that accounts for dispersion interactions either implicitly or through corrections when studying the interaction between hydrogen and IRMOF-1 . The most accurate stabilization energies of the adsorbed hydrogen have been determined using ωB97XD/cc-pVDZ-PP at the optimized configuration from ωB97XD/LANL2DZ. The M06-2X/LANL2DZ method is a good approach to search H2 adsorbed to each of the surface sites were first optimized within the IRMOF-1 super-cell cluster. This model system allows for the comparison of H2 stabilization energies in the extended crystal framework relative to the fragment based models, as well as the study of perturbations in the stabilization energies that may occur due to site-to-site interactions between H2 molecules at higher loading. The twelve optimized H2 configurations in the super-cell are illustrated in 2 on the α site, α(||)1–4, while only one perpendicular configuration was optimized α(⏊). Five hydrogen configurations were also optimized on the β site, one parallel, β(||), which is predicted to have the smallest stabilization energy among the β site configurations, while the others, β(⏊)1–4, are the minima on the perpendicular configuration potential energy surface. Two configurations were optimized at the γ site. Note that a third configuration has been previously reported at the γ site, termed III(B) in reference [2 configurations on the δ and ε sites were optimized and these optimized H2 configurations are shown in 1–4 wherein the hydrogen molecule lies on a plane that is parallel to the phenylene plane. The most stable parallel hydrogen, δ(||)1, lies over the y axis 1, in which the hydrogen molecule is perpendicular to the phenylene plane. The next stable configuration is the ε(||) in which the hydrogen is on the phenylene plane, and parallel to a phenylene edge. The third configuration is the ε(⏊)2, in which the hydrogen is on the phenylene plane, but perpendicular to a phenylene edge.The individual configurations of Heference , however2 in IRMOF-1 super-cell, model fragments with and without BSSE corrections are listed in 1 is 0.04 kJ/mol lower than that of ε(||), however their energy order reverses in model frag2, such that ε(⏊)1 has a 0.17 kJ/mol higher SE value than ε(||). Similar behavior is observed using ωB97XD. The SE values of δ(⏊) and δ(||)1 also change significantly when using a super-cell. In the M06-2X approach, the SE difference between δ(⏊) and δ(||)1 configurations increases from 0.03 to 0.09 kJ/mol when the investigated system changes from the model frag2 to the super-cell. These data provide an initial assessment of the energetic impact caused by long-range affects induced within the crystal lattice relative to the model fragments, a feature that has only been previously inferred by comparing the stabilization energies of different sites as a function of the size of different fragment models [The stabilization energies of all configurations in the super-cell are listed in t models .2. The initial 64 hydrogen molecules were placed in the super-cell cluster at the most stable H2 configuration obtained from optimization of each individual site in isolation. All 64 positions were then simultaneously optimized using M06-2X/LANL2DZ. The coordinates of super-cell IRMOF-1 and the coordinates of 64 H2 adsorbed at optimized positions are listed in 2 on each site was then calculated after the optimization of highly H2 loaded system. The new SE values are listed in 2/cell) and 2 configurations (1 H2/cell) and with all positions filled (64 H2/cell). The SE values of the γ site is stabilized by 1.4 kJ/mol when in the presence of the other H2 molecules. The change of SE value indicates non-negligible intermolecular interactions among these hydrogen molecules, a feature explored by NBO analysis [γ + Hγ) bonding orbital of the γ-bound H2 donates into the anti-bonding orbital of α–bound hydrogen, σ*(Hα–Hα). Here the Hγ and Hα are the 1s atomic orbitals of the γ– and α–bound hydrogen molecules, respectively. Representative pictures of the natural bonding and anti-bonding orbitals are plotted in 2 is ~0.79 kJ/mol. The optimized distance between donor hydrogen and acceptor hydrogen atoms is 3.21 Å.The effect of intermolecular interactions between sorbate molecules has been investigated by populating all the primary sites with adsorbed Hanalysis . All pos2 molecules at the γ and α positions were studied along the potential energy surface where the donor hydrogen molecule was moved toward the center of the super-cell and all other atoms and H2 were kept fixed. The PES scan began at a distance of 3.98 Å and ended at 3.58 Å. The interaction energies were first calculated with M06-2X/LANL2DZ, and then using HF with LANL2DZ as a comparison. Thus, the MP2 orbital interaction energies are examined using the NBOs from two methods, one capable of describing dispersion and charge transfer (M06-2X), and one that cannot capture the essential physics of dispersion, but does describe charge-transfer rather well (HF). The interaction energies between donor and acceptor H2 molecules with respect to the distance between these H2 molecules are given in i,j . These second-order energies are generally proportional to the square of the orbital overlap. As the NBOs are orthogonal, the overlap of the pre-orthogonal NBOs  has also been examined, and in presented in 2, and α–H2 participate in site-to-site charge transfer interactions that significantly stabilize H2 adsorption. Importantly, we confirm that this interaction does not only exist at the theoretical maximum loading of 64 adsorbed H2 within the super cell. Indeed, merely calculating two isolate H2 molecules at the geometry fixed within the super-cell using NBO leads to an interaction energy of ~0.46 kJ/mol.NBO is not traditionally employed to study weak intermolecular interactions, and as such it may be possible that the charge transfer is merely a manifestation of enhanced dispersion interactions that should occur at high loading. In this work, NBO analysis is preferred to study charge transfer over Symmetry-Adapted Perturbation Theory (SAPT) because charge transfer is enclosed partially in other energy terms of SAPT instead of being explicitly stated . To furt1 >> β(⏊)1 > γ(⏊) > δ(⏊) > ε(⏊)1, consistent with previous studies. To investigate the possible interaction among adsorbed hydrogen molecules at high pressures, 64 H2 molecules sorbed on the five primacy sites were populated and optimized simultaneously. This sorption configuration represents a theoretical maximum with all 20 identified site types populated. Though this configuration (14 wt %) has not been achieved experimentally (11.5 wt %), it does represent a suitable model system to explore neighboring adsorbate interactions. The high hydrogen concentration significantly stabilizes the γ site. Natural bond order analysis reveals that delocalization of electrons, or charge transfer, from the σ orbitals of the H2 bound at the γ positions into the σ* orbitals of H2 bound at the α sites leads to stabilization of the interaction of H2 at the γ by 1.4 kJ/mol, respectively (using M06-2X/LANL2DZ). Indeed, independent NBO calculation of only the two adsorbed α- and γ-bound H2 at the same geometries as in in the super cell yields similar observations. Given that the distance between these neighboring H2 molecules is only 3.21 Å, their site-to-site interactions should not be ignored in future computational studies at modest to high H2 pressures, and point to a general consideration of loading when examining the adsorption of weakly bound small molecules to porous media.Twenty hydrogen configurations on five primary sites were optimized using M06-2X/LANL2DZ in a super-cell consisting both of an edge and face cube sub-cells of IRMOF-1 (MOF-5). Their stabilization energy order is α(||)