The spine is the central skeletal support structure in vertebrates consisting of repeated units of bone, the vertebrae, separated by intervertebral discs (IVDs) that enable the movement of the spine. Spinal pathologies such as idiopathic back pain, vertebral compression fractures and IVD failure affect millions of people worldwide. Animal models can help us to understand the disease process, and zebrafish are increasingly used as they are highly genetically tractable, their spines are axially loaded like humans, and they show similar pathologies to humans during ageing. However, biomechanical models for the zebrafish are largely lacking. Here, we describe the results of loading intact zebrafish spinal motion segments on a material testing stage within a micro-computed tomography machine. We show that vertebrae and their arches show predictable patterns of deformation prior to their ultimate failure, in a pattern dependent on their position within the segment. We further show using geometric morphometrics which regions of the vertebra deform the most during loading, and that finite-element models of the trunk subjected reflect the real patterns of deformation and strain seen during loading and can therefore be used as a predictive model for biomechanical performance. Back pain and IVD degeneration affect millions of people worldwide ,2, and va), their spine is antero-posteriorly loaded as a result of swimming through viscous water [Animal models, typically rodents, are frequently used to study mechanisms of spinal pathology . Howeverfigure 1us water . Zebrafius water , and devus water  and ageius water . Howeverex vivo [Finite-element analysis (FEA) has proven a pivotal tool in the study of biomechanical subjects , and offex vivo ,11. FEA ex vivo ,13 and tex vivo .ex vivo.Here, we describe a novel integrated experimental platform that brings together imaging, modelling and real-world validation to explore the biomechanics of intact zebrafish spinal MSs. We generated an FEA model of the spine, which we validated with a loading experiment using a high-precision material testing stage (MTS) under set loading regimes using micro-computed tomography (µCT). Three-dimensional geometric morphometrics (3D-GM) was used to explore patterns of deformation seen in each vertebra during loading. Comparison of results demonstrated that our FEA model accurately predicted the relative patterns of deformation and strain experienced by real samples loaded 2.2.1.a–c).One-year-old, wild-type (WT) zebrafish were fixed in 4% paraformaldehyde and dehydrated to 70% EtOH. MSs were acquired by making two cuts in the trunk, between the morphologically homogeneous vertebrae 18 and 24 of a total of 33 vertebrae  figure a–c.2.2.n = 3) was performed and if required, vertebrae were trimmed to retain three complete vertebrae and associated IVDs (b–d). Samples were stabilized (anterior-up) in the MTS2 using cyanoacrylate glue. The MTS2 was programmed to perform a sequential series of seven scans at a series of increasing loads  in a Bruker SKYSCAN 1272 µCT system. Radiographic visualization of each MS .An MS surface mesh was created based on a 1-year-old WT specimen µCT scanned using a Nikon XTH 225ST μCT system as described under two conditions: (a) native state and (b) contrast-enhanced following 14 day incubation in 2.5% phosphomolybdemic acid . Scan . Deformed meshes from each step were exported as surface files and analysed using 3D-GM for quantitative comparison between relative and absolute patterns of deformation predicted by FEA and observed in MTS data.The complete vertebral surface mesh was imported into Simpleware ScanIP  to create an FE model. The model consisted of 1 054 187 linear tetrahedral elements joined at 257 392 nodes comprising four material types: vertebral bone, annulus-fibrosus, nucleus-pulposus and stainless steel (figure 12.4.a–c) linked by 300 surface sliding semi-landmarks (using the ‘buildtemplate’ function). By assigning the same landmarks in each scan (using the ‘digitsurface’ function), we compared the first scan with subsequent scans of the same vertebra using generalized Procrustes analysis . Resulting shape variables were subjected to principal component analysis (PCA) to identify the principal patterns of variation between scans of the same vertebra, and isolate trends in deformation with increasing compressive load.Three-dimensional geometric morphometrics analysis of vertebral deformation was performed using the ‘Geomorph’ package for the R statistics software . For eacfigure 23.3.1.j,l). All samples failed between 12 and 16 N upon shallow angle fracture in the central vertebra, with the smallest specimen (specimen 3) failing at the lowest force . This is higher than maximum aquatic forces experienced during swim training by Fiaz et al. [figure 1g), suggesting the IVD acts like a coiled spring that may further contribute to the ultimate strain and failure of the segment when released via small-scale bone fracture (figure 1h). The surrounding epaxial musculature showed no obvious deformation or damage until the entire MS failed, at which point muscle fibre organization was lost (figure 1i–l). Comparison between MTS data and FEA results demonstrated strong spatial correlation between maximum predicted strain and ultimate point of failure in the central vertebra (figure 1m–o).To test the range of compressive loads that the MS could resist until failure, we subjected an MS to exponentially increasing compressive forces from 1 to 100 N. This specimen failed at 16 N whereupon the central vertebra fractured mid-centrum. A primary loading regime between 1 and 16 N was thus established  for the figure 1z et al. , which r3.2.d,i,n), showing consistent dorsoventral compression, and lateral compression that is reversed at higher loads potentially due to elastic rebound of the IVD and fracturing along the zygopophyses that occurs at these loads . Comparison with FEA data  suggests that the FE model accurately predicts these patterns , and that patterns of deformation could explain the first signs of damage prior to failure. In both datasets, the anterior vertebra undergoes most deformation, particularly posterior deformation of the arches (figure 2e–h). The central vertebrae and arches show strong torsion (figure 2j–m), increasing through the loading regime leading to the failure of the segment . The posterior vertebra shows the least deformation and is most isotropic in pattern (figure 2o–r), potentially due to protection offered by the anterior IVDs.We found characteristic patterns of deformation and strain in response to compressive loading of zebrafish vertebrae. Three-dimensional results from MTS data follow distinct trends for each vertebrae between the three specimens d,i,n, shse loads . This re segment l,o. The ex vivo loading of vertebral MSs validates the accuracy of our FEA model for predicting patterns of deformation and strain across these structures. This offers a step towards a digital ‘sandbox’ approach to modelling the effects of genetic, physiological and morphological properties on the reaction and resistance of vertebral MSs to loading. Inputting specific properties of vertebral samples into a validated FE model will allow their effects on the biomechanics of the spine to be quantitatively tested in silico, allowing the relative contributions of shape and material properties to be explored and empirically tested. This will aid comparison of mechanical performance between different model systems. As an advantage of the zebrafish system is the wealth of mutants modelling human disease genetics [Comparison with genetics , compari


Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that is overexpressed and activated in many human cancers. FAK transmits signals to a wide range of targets through both kinase-dependant and independent mechanism thereby playing essential roles in cell survival, proliferation, migration and invasion. In the past years, small molecules that inhibit FAK kinase function have been developed and show reduced cancer progression and metastasis in several preclinical models. Clinical trials have been conducted and these molecules display limited adverse effect in patients. FAK contain multiple functional domains and thus exhibit both important scaffolding functions. In this review, we describe the major FAK interactions relevant in cancer signalling and discuss how such knowledge provide rational for the development of Protein-Protein Interactions (PPI) inhibitors. During the last two decades, considerable efforts have been made in the understanding of the molecular mechanisms of diseases. However, at present, only a small portion of proteins (around 400) involved in diseases have been explored as therapeutic targets . Indeed,2) compared with those involved in small-molecule-protein interactions (300–1000 Å2) [3 in contrast to PPI, which involve an average of six small pockets with a pocket volume around 54 Å3 [Among these potential new targets, protein-protein interactions (PPIs) are of considerable and growing interest. Indeed, PPIs are critical for many aspects of cell biology and are involved in physiological as well as pathological processes. However, targeting PPIs with small “drug-like” molecules is challenging for at least two main reasons. First, the contact surfaces involved in PPI are large (1500–3000 Å1000 Å2) ,5. In PPnd 54 Å3 . It is tDespite these challenges, several evidences provide hope for discovering small drug-like molecules that would target PPI interfaces. Even though the protein-protein interfaces are large, mutational studies have shown that a small subset of residues involved is essential for high-affinity binding; they are called hot spots . The disSuccessful examples of PPI inhibitors have been reported in distinct biological pathways. One of those and probably the best-characterized are the inhibitors of the interaction between p53 and human protein double minute 2 (HDM2). MDM2 (the mouse homologue of HDM2), which is an ubiquitin E3 ligase was initially found to binds to the tumour suppression protein p53 leading to its proteasomal degradation . p53 is Another example of PPI inhibitors is small inhibitors of the Bcl-2 family in clinical trials especially for the treatment of patients with lymphoid malignancies. The Bcl-2 (B-cell lymphoma 2) family contains important regulators of apoptosis. These proteins govern mitochondrial outer-membrane permeabilization and form homodimers or heterodimers with other family members to mediate either a pro-apoptotic or a pro-survival signal. The effect of pro-apoptotic proteins Bak and Bax are blocked by the interaction with anti-apoptotic partners such as Bcl-2 and Bcl-X. This interaction is mediated by a hydrophobic BH3 domain in pro-apoptotic protein containing four key hydrophobic residues  . DisruptThus, PPI inhibitors may represent an interesting alternative to target protein kinases. Indeed, the majority of reported protein kinase inhibitors are ATP-binding competitors which bind to the active kinase conformation (type I inhibitors) resulting in often limited kinase selectivity due to the high degree of conservation within the ATP-site. Searching for competitive inhibitors rather than ATP-competitive inhibitors may be a more rational approach. At present, more than 1500 ATP-dependent enzymes have been identified with 420 Ser/Thr kinases and 90 Tyr Kinases. Moreover, the high intracellular ATP concentrations necessitate high intracellular concentrations (μM) of the inhibitor to effectively inhibit the target, even when the affinity of the inhibitor is in the nanomolar range . On the fak deletion in the epidermis [More than twenty years ago, FAK was first described as a substrate of the oncogene product of the sarcoma virus (v-Src) and as a protein highly phosphorylated upon clustering of Integrins at focal adhesions ,27,28. Spidermis . This prNevertheless, besides its kinase function, FAK possess also scaffolding functions that are highly relevant in cancer signalling . Indeed,2 [The best characterized mechanism that promotes FAK activation involves Integrin receptor clustering upon cell binding to the extracellular matrix which has been shown to involve binding of the β Integrin cytoplasmic domain to FAK ,36,37. F2  the tetr2  and grow2 ,43,44,452 . On the 2 . Consequ2 . Early e2 .216AKTLRK located in the FERM F2 lobe [Using a FRET-based biosensor for monitoring FAK activation it was also confirmed that PIP2 triggers a change in FAK conformation state which likely represents unbinding of the FERM-kinase domain . However F2 lobe . The FER F2 lobe . This re F2 lobe . Latter  F2 lobe .Other mechanisms of FAK autoinhibition have also been unveiled. First, based on early structural data on the FERM domain together with different linker segments, the Tyr397 site was shown to be inaccessible because of an interaction with the F1 lobe of the FERM domain and inhibition of Src binding due to sequestration of the SH3 binding site via an intramolecular interaction with the F3 lobe . Second,Several mechanisms seem to be implicated in the control of wound-induced cell polarity. Indeed, it was first described that FAK directly interact and phosphorylate the actin regulatory protein N-WASP thereby promoting cell migration . Integri216KAKTLRK222 basic patch which mediates interaction with other proteins, another partially overlapping surface exposed basic patch involving K190/191, K216/218, R221 and K222 has been shown to mediate FAK localisation to the nucleus. This raised the intriguing possibility that in the absence of FAK activation signal involving binding of c-Met, PIP2 or other molecules to these basic residues, FAK will be mainly located to the nucleus due to unmasking of the NLS signal. Besides, another basic patch namely R177/178 located in the F2 lobe is also consistent with NLS signal activity [The FERM domain of FAK contain different elements such as nuclear localization signals (NLS) and nuclear export signals (NES) that regulate the shuttling of FAK between the nucleus and FA. In the F2 lobe, beside the activity . Interesactivity . SumoylaNormal cells require adhesion to the ECM in order to survive and grow a process that is dependent on FAK activation. Indeed, many reports have shown that inhibition of FAK leads to the onset of apoptosis ,74. BesiThe search for inhibitors of PPIs can be made by selectively targeting mechanisms that modulate the kinase activity or specific interactions with known regulators of cancer-related pathways. For the first purpose, one should target protein-protein interactions that play a role in the regulation of kinase activity in order to achieve targeting specificity. In this aim, a protein pharmacophore model for the FAK-related kinase Pyk2 F3 FERM domain was recently generated and served as a template for the screening of the LeadQuest database. This led to the identification of a small compound that directly bind to the Pyk2 FERM domain and inhibited the Pyk2-stimulated glioma cell migration . NeverthAnother successful example came from the same group targeting the FAK-Mdm-2 interaction. Mdm-2 is a p53 target involved in p53 proteasomal degradation which was found to bind FAK via the F3 lobe of the FERM domain . Using tThe interaction between FAK and IGF-1R was also partially characterized. It appears that this interaction encompasses FAK amino-acid 127–243 and the N-terminal part of IGF-1R . Using tTyr397, which is located between the FERM and the kinase domain, is the major FAK activation site . FAK phoFAK phosphorylation at Tyr397 and FAK activation is also important in tumour-associated endothelial cells where it regulates endothelial cell motility, angiogenesis, endothelial and cell sprouting ,107,108.The majority of FAK binding partners at the kinase domain involved the binding of SH2-containing proteins to FAK phosphorylated at Tyr397 which includes Src  but also−/− cells have shown increased Pyk2 activity due to compensatory effect. However, several reports suggest that although FAK kinase activity is necessary for cell motility, it may be not essential for cell proliferation and survival. Indeed, as already mentioned, these later effects are most certainly mediated by the FERM domain of FAK through regulation of p53 and the Receptor-Interaction Protein.FAK phosphorylation and activation drives many cancer-related processes. Therefore, based on this knowledge, two strategies can be developed to inhibit FAK function in cancer cells. The first is to inhibit the kinase function of FAK and the second to alter Src binding to FAK because this event is at the basis of the full FAK kinase activity. Pharmacological companies like Novartis or Pfizer have developed several kinase inhibitors of FAK which are almost all small ATP-competitive molecules. One of the first compounds developed was TAE-226 which exhibit nanomolar inhibitory activity toward FAK but inhibit also insulin-like growth factor-I receptor kinase ,114. In in-vitro, Y11 dose-dependently decreased viability, adhesion and clonogenicity in colon and breast cancer cell lines while significantly reducing tumour growth in a colon cancer cell mouse xenograft model [To avoid selectivity problems typically found with competitive inhibitors of the ATP binding site, inhibiting the scaffolding function of FAK with the use of PPI inhibitors can be an attractive alternative approach. Specific interactions between SH2 domain-containing proteins and their phosphotyrosine counterparts play a significant role in tyrosine kinase signalling pathways. Thus, blocking the interaction between SH2 domain and its binding protein may inhibit overactive signalling pathways in cancer cells. For example, using structure-based virtual screening of the National Cancer Institute chemical libraries, small inhibitors of Stat3, targeting the Stat3 SH2 domain bound to a Stat3 phosphotyrosine peptide, were discovered . In the ft model . Moreoveft model ,127. FinFAK contains three prolin-rich (PR) domains mediating many protein interactions : PR1 locCortactin is another important protein that directly interacts with both FAK PR2 and PR3 domains thereby being phosphorylated at its tyrosine sites Tyr 421 and Tyr 466 which leads to FA dynamic regulation . CortactThe shuttling of FAK in and away from FAs is crucial for FAK regulation of the migration/invasion pathway driven in part by the spatial-temporal control of Src activity. In cancer cells, inhibition of FAK leads to altered Src trafficking via at least two major pathways. The first implies a relocation of Src from FAs to invadopodia through its interaction with the FAK homologue Pyk2 leading to enhanced matrix degradation . The secOther SH3-containing domains that interact with FAK involve critical regulators of the Rho-GTPase family, which are important molecular switches that regulate cell motility. GTPase activating proteins (GAP) and guanine-nucleotide exchange factors (GEF) bind to these domains to regulate Rho, Rac or Cdc42. For example, the Rho regulator, implicated in cytoskeleton reorganisation, termed PSGAP (Pleckstrin homology and the SH3 domain containing rhoGAP) has been shown to also interact with FAK. This protein binds to a sequence located near Pro 859 in Pyk2 close to the FAT domain and presumably to the equivalent PR3 sequence in the FAK structure, although this has not been formally demonstrated . The GraOn the other hand, FAK associates with the guanine nucleotide exchange factor PDZ-RhoGEF to modulate Rho/Rho kinase II signalling necessary for focal adhesion dynamic at the tail and trailing edge retraction but the structural determinants of FAK required for this interaction were not described . Both PDThe effects induced by the phosphorylation of FAK at Ser 732, that is close to PR2 have been addressed in several cell types. The first reports describe that FAK phosphorylation at this site is important for microtubule organization and microtubule-dependant mechanisms such as nuclear translocation, mitosis and neural cell migration ,152. LatAt the moment, no PPI inhibitors targeting FAK interactions via its PR domains has been developed. However, this strategy may be valuable as protein domains that bind proline-rich motifs are frequently involved in signalling events. Indeed, for FAK, P878A/P881A mutation were associated to decrease expression levels of markers for epithelial-mesenchymal transition in vivo in a mouse model of human breast cancer which correlate with an inhibition of the FAK/Endophilin A2 interaction . Thus, tCAS [in-vitro association of Talin with the Integrin β1 cytoplasmic domain has been detected, it was suggested that Talin may target FAK to FAs [2 at sites of Integrin activation thereby connecting FAK to nascent adhesions [The C-terminal domain of FAK interacts with several FA-associated proteins including Paxillin and Talin ,159,160,CAS ,161, GrbCAS , the p85CAS  and VEGFCAS . FurtherCAS ,165,166 K to FAs . StudiesK to FAs ,169. MorK to FAs . Alternadhesions . Other pdhesions  and the dhesions . Interacdhesions .One important motif of the FAT domain is the Tyr925 phosphorylation site, which has been identified as a Src-dependent process because its phosphorylation is significantly reduced in cells expressing a kinase-defective mutant of Src . Upon phCell motility also requires the coordinated turnover of nascent adhesions at the leading edge and the disassembly of FA at the rear, a process dependent upon FAK phosphorylation at Tyr925. Because phosphorylation of Tyr925 and subsequent binding of SH2-containing protein require rearrangement of the FAT structure, it has been suggested that this process would lead to alteration of FAK-Paxillin interactions thus ultimately leading to FAK removal from FA . HoweverEarly studies have shown that agonists of G protein-coupled receptors, including Bombesin and lysophosphatidic acid stimulate FAK phosphorylation at Ser910, through an ERK-dependent pathway. Moreover, growth factors that stimulate the ERK pathway in 3T3 fibroblast, like FGF, phosphorylate FAK at Ser910 whereas others like Insulin which is unable to simulate the ERK pathway does not promote FAK phosphorylation at Ser910 further indicating the requirement of ERK for phosphorylation at this site ,183. IntL1034S, which destabilizes the four-helix bundle of the FAT domain, disrupts FAK interaction with p190RhoGEF thus clearly indicating the requirement for an intact FAT domain [We described previously interactions between Rho-GTPase family regulators with proline-rich domains of FAK but FAK also interacts via its FAT domain with some of these regulators. First, as discussed before, FAK interacts directly or via formation of a complex that include p130Cas, with Dock180, a GEF for Rac thereby promoting lamellipodia formation. FAK also regulate Rho via interaction with p190RhoGEF that stimulate Rho activity. Although the interaction domains were not precisely mapped, mutation of FAKT domain . AnotherT domain . GIT1 isT domain . NeverthT domain . On the T domain . AnotherT domain . This inFAK localisation at FA is essential for its regulation by Integrin signalling as demonstrated using replacement of the FAT sequence of FAK . HistoriSuccessful examples have now proven the efficiency of PPI inhibitors as some of these already reached clinical trials. FAK is at the crossroads of integrin and growth factors signalling and, as FAK is overexpressed in many human cancers, FAK is frequently associated with the promotion of oncogenic signals and the inhibition of tumour suppressive pathways. This makes FAK a clearly interesting cancer target. The development of PPI inhibitors targeting FAK is thus quite promising as FAK, via several specific subdomains and numerous sites of phosphorylation, is at the heart of an important interactome. Several small molecules targeting FAK kinase function are currently being developed with some success in clinical trials. Especially, targeting FAK in combination with therapies against other signalling pathways may be a promising approach for the treatment of drug-resistant cancers. Therefore, with the mapping of many FAK interactions at the molecular level, the search for inhibitors of FAK scaffolding function may lead to new promising therapeutics.


The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in the regulation of cell signaling, proliferation, survival, migration and vesicle trafficking in normal cells and is frequently deregulated in many cancers. The p85α protein is the most characterized regulatory subunit of the class IA PI3Ks, best known for its regulation of the p110-PI3K catalytic subunit. In this review, we will discuss the impact of p85α mutations or alterations in expression levels on the proteins p85α is known to bind and regulate. We will focus on alterations within the N-terminal half of p85α that primarily regulate Rab5 and some members of the Rho-family of GTPases, as well as those that regulate PTEN (phosphatase and tensin homologue deleted on chromosome 10), the enzyme that directly counteracts PI3K signaling. We highlight recent data, mapping the interaction surfaces of the PTEN–p85α breakpoint cluster region homology (BH) domain, which sheds new light on key residues in both proteins. As a multifunctional protein that binds and regulates many different proteins, p85α mutations at different sites have different impacts in cancer and would necessarily require distinct treatment strategies to be effective. PIK3R1 , PIK3R2 (p85α) and PIK3R3 (p55α). All the isoforms contain a pair of SH2 (Src homology 2) domains , flanking an interSH2 (iSH2) domain. These C-terminal domains are responsible for mediating binding to upstream tyrosine phosphorylated sites on receptors or adapter proteins, and to the p110-PI3K protein. In addition to the C-terminal domains, the larger p85 isoforms contain N-terminal regions that include: an SH3 (Src homology 3) domain and a breakpoint cluster region homology (BH) domain that has GTPase activating protein (GAP) activity (Class IA phosphatidylinositol 3-kinase (PI3Ks) consist of an 85 kDa regulatory subunit (p85), partnered with a 110 kDa catalytic subunit (p110). The p85 isoforms are encoded by three genes, activity . These r2) to generate phosphatidylinositol 3,4,5-trisphosphate P3), a key signaling lipid.The p85α protein has a well-characterized role in binding, stabilizing and regulating the activity of the p110α catalytic subunit of PI3K ,3. Tyros3 formation recruits pleckstrin homology domain containing proteins, including PDK1 (phosphoinositide-dependent protein kinase-1) and Akt to the membrane, facilitating the phosphorylation and activation of downstream Akt signaling ,31PIK3CAhenotype  , glioblastoma , colorectal  and prostate   b 30,31],31PIK3R1%, 4/70) b. Mutati29%, 172/86, gliobctal 6%, /594 and Rho GTPases are signaling G protein members of the Ras superfamily and include Rho, Rac and Cdc42. These small GTPases function to regulate the organization of the actin cytoskeleton and focal adhesions, which are important factors influencing cell migration and invasion . Small G3 and the subsequent sustained activation of Rac-GEFs, including Vav1 and Sos1, both of which require PIP3 for their activity ,31PTEN l124/564) c rendersPTEN and PIK3R1 also include a high frequency of deletions  [The vast majority of cancer-associated alterations are of low frequency and remain variants of unknown significance . This reokb.org) . This inokb.org) . The scaokb.org) . These rokb.org) , more muThe discovery of cancer driver mutations does not effectively predict therapeutic benefit in the absence of additional information. Computational analyses have been used in conjunction with tools such as protein structure data to define colocalized mutations within the folded protein . NetworkEGFR, PIK3CA, BRAF and ERBB2), as well as potentially druggable genes . Two cell lines were used for each target to determine the growth factor-independent cell viability when the mutant was expressed, as compared to the wild type control. A functional proteomic analysis was also carried out on a subset of these cell lines using a reverse phase protein array (RPPA) to quantify target protein levels and specific post-translational modifications [http://bioinformatics.mdanderson.org/main/FASMIC) was also developed to ensure the data were broadly available [Systematic functional characterization of cancer-associated mutations is a huge challenge, but is essential for implementing precision oncology. A recent report describes a moderate-throughput functional genomics platform and uses it to annotate >1000 cancer variants of unknown significance . This stications . Resultsvailable . These tMutations in different regions of p85α, even within the same domain, can exert very different cellular effects depending on which of the many p85α binding partners that they impact . The fac


In vitro kinase assays showed that PKD, a COP9 signalosome-associated-kinase, is able to phosphorylate Sos1. The intracellular levels of Sos1 protein were clearly diminished following CSN3 or PKD knockdown. A sizable fraction of the endogenous Sos1 protein was found ubiquitinated in different mammalian cell types. A significant reduction of RasGTP formation upon growth factor stimulation was also observed in CSN3-silenced as compared with control cells. Our data suggest that the interaction of Sos1 with the COP9 signalosome and PKD plays a significant role in maintenance of cellular Sos1 protein stability and homeostasis under physiological conditions and raises the possibility of considering the CSN/PKD complex as a potential target for design of novel therapeutic drugs.Sos1 is an universal, widely expressed Ras guanine nucleotide-exchange factor (RasGEF) in eukaryotic cells. Its N-terminal HD motif is known to be involved in allosteric regulation of Sos1 GEF activity through intramolecular interaction with the neighboring PH domain. Here, we searched for other cellular proteins also able to interact productively with the Sos1 HD domain. Using a yeast two-hybrid system, we identified the interaction between the Sos1 HD region and CSN3, the third component of the COP9 signalosome, a conserved, multi-subunit protein complex that functions in the ubiquitin–proteasome pathway to control degradation of many cellular proteins. The interaction of CSN3 with the HD of Sos1 was confirmed in vitro by GST pull-down assays using truncated mutants and reproduced in vivo by co-immunoprecipitation with the endogenous, full-length cellular Sos1 protein They are continuously cycling between inactive (Ras-GDP) and active (Ras-GTP) conformations through a process modulated by negative  and positive  regulators. The Sos1 and Sos2 proteins constitute the most universal and widely expressed family of RasGEFs in mammalian cells8 exhibit a multi-modular structure featuring conserved distribution of specific, functional domains along their amino-, central-, or carboxy-terminal regions6. The carboxyl-terminal end of Sos proteins is a proline-rich (PR) region adopting left-handed type II helix conformation11, which binds the SH3 domains of Grb213. Two distinct hSos1 isoforms differing in their Grb2-binding ability and biological potency have been identified in this region15. The amino-terminal region of Sos proteins includes the histone-like domain (HD), Dbl-homology domain (DH), pleckstrin domain (PH), and helical linker (HL)6. The Ras-Exchange motif (REM) and the CDC25-homology (CDC25-H) domains are located centrally, between the HL domain and the PR motif, and constitute the catalytic-center of the nucleotide-exchange activity on Ras proteins16. Interaction of the REM domain with the switch-2 region of Ras mediates binding to Ras-GDP whereas, simultaneously, connection of the two β-sheets of the CDC25H domain with the switch-1 region of Ras promotes dissociation of GDP17.The highly homologous, ubiquitously expressed Sos1 and Sos2 proteins19. This N-terminal region concentrates most of the activating (gain-of-function) Sos1 germline mutations detected in Noonan syndrome cells21, where hyperactivation of Ras is the result of reduced Ras-GEF self-inhibition6. The HD region shows high homology to histone H2A and contains two tandem histone folds19. Structural studies have shown that the HD stabilizes the self-inhibited conformation of Sos proteins through its basal conformational interaction with the DH-PH region22. In this conformation, the DH-PH domain blocks the Ras allosteric binding site22 and, only after the stimulation by growth factors, the complete activation of Sos may proceed. This activation requires growth-factor-induced generation of phosphatidic acid via phospholipase D223 followed by recruitment of Sos to the plasma membrane through the PH domain6 and HD binding to phosphatidic acid, thus promoting Sos RasGEF activity in the plasma membrane24.Various structural studies indicate that the amino-terminal region plays a role in allosterical regulation of the overall RasGEF activity of Sos25 and also show experimental evidences supporting the functional relevance of this HD-mediated interaction, indicating that the COP9 signalosome and its associated PKD contribute to regulating the half-life and homeostasis of cellular Sos1 protein and therefore the activity of the Ras signaling pathway.Based on the HD tertiary structure and its function as a modulator of Sos-GEF activity, we hypothesized that this domain could interact in vivo with some cellular proteins in order to regulate Sos RasGEF overall functionality. Here, we demonstrate in vitro and in vivo interaction between the HD of Sos1 and CSN3, a subunit of the highly conserved COP9 signalosome complexTo identify candidate cellular proteins able to interact with the amino-terminal region of hSos1, we performed yeast two-hybrid screens using a host strain  that was co-transformed with a HeLa cDNA plasmid library (clones fused to B42-activation domain) and different plasmid baits coding for various truncated fragments of the N-terminal region of hSos1 (fused to LexA-DNA-binding domain) Fig. . As inteCOP9 signalosome complex subunit 3 protein (CSN3) (NP_001303284.1).Initial screening using a LexA-NDP plasmid bait coding for the first 548 amino-terminal residues of hSos1 yielded high numbers of positive, interacting clones. After successive rounds of screening, two clones showing positive interactions were selected for further study Fig. . After D− plates   of the NDP region Fig. . As intetes Fig. . In conttes Fig.  and grewium Fig. . Judgingide Fig. . In contide Fig.  and did tes Fig. . The spe3]) Fig. .These observations in a two-hybrid system strongly suggest that cellular CSN3 is able to bind to the amino-terminal region of hSos1, and that this molecular interaction specifically involves the HD region/domain of Sos1 protein.To directly validate our yeast two-hybrid findings and confirm the ability of CSN3 to bind to different hSos1 N-terminal fragments/domains, we carried out in vitro binding experiments using pull-down assays. Cytoplasmic extracts from HeLa cells were incubated with purified GST-HD, GST-DH-PH, and GST-NDP fusion proteins coupled to glutathione–sepharose beads that were subsequently tested by means of immunoblotting with antibodies against CSN3 Fig. . Whereas26 and, in view of our two-hybrid data and in vitro binding observations, we wished to ascertain if the interaction between the N-terminal region of Sos1 (NDP peptide) and the COP9 signalosome occurs also in vivo, and the status of cell stimulation under which such an interaction may occur.CSN3 is a component of the multi-protein cellular COP9 signalosome complex27 and therefore this approach would not compromise any potential, putative CSN3-Sos1 interaction. These WB data clearly demonstrated the interaction of the COP9 signalosome with the endogenously overexpressed HA-NDP peptide in both serum-starved or serum-stimulated HeLa cells  NDP construct. Direct cytoplasmic lysates, or immunoprecipitates thereof using polyclonal rabbit anti-CSN7a antibody or control unspecific IgG, were subsequently probed by immunoblotting with anti-HA antibodies Fig. . Notice lls Fig. .28 so as to exclude any potential interference by the highly homologous Sos2 protein. Cultures of these MEFs were starved before FCS stimulation and then direct cellular lysates or immunoprecipitates of the same lysates using anti-CSN7 antibodies were probed by WB immunoblotting with anti-Sos1 antibodies  or human HeLa cells Fig. , as well29, we used immunoblot assays to look for this possible postranslational modification of Sos1 in primary, WT or Sos1-depleted MEFs32. Our WB data showed that a sizeable portion of the endogenous Sos1 was found ubiquitinated in the lysates of primary MEFs growing under normal conditions in culture . As ubiquitination is central to the UPS in order to generate degradation signals on substrates destined for destructionure Fig. .35.The COP9 signalosome (CSN) is a conserved protein complex with isopeptidase activity that controls eukaryotic protein degradation through the UPS by regulating the activity of cullin-RING ligase (CRL) families of ubiquitin E3 complexes. Multiple reports document the involvement of CSN in different cellular and developmental processes, including DNA-damage responses or control of gene expression and the cell cycles cells were incubated in absence or presence or cycloheximide (CHX) for up to 9 h, and the decay of the Sos1 protein was followed over time by WB immunoblotting using anti-Sos1 antibody  or HD-truncated (AU5-hSos1-ΔHD) forms of human Sos1 proteins were used as substrates for in vitro kinase assays with PKD-KD (kinase dead) and PKD-CA (constitutively active) mutants of PKD  was reported to be associated to the COP9 signalosome complexPKD Fig. . Our assPKD Fig. . Consistorm Fig. .Fig. 4PK42 are located within various regions of Sos1, including the REM and CDC25H motifs  or the DH region (imperfect matches at positions S385 and S401). Given the known conformational interactions between the HD and the DH-PH motifs, it will be important to test in future the phosphorylation status of individual mutations at each of those positions to determine the exact mechanistic contribution of their potential phosphorylation to cellular Sos1 stability.Potential matches for the consensus substrate target motif (L/I-X-R-X-X-S/T) recognized by PKD27, we wished to explore further the potential in vivo relationship between PDK activity and cellular Sos1 protein homeostasis.As PKD1 is able to phosphorylate the Sos1 protein in vitro and is also known to associate with the COP9 signalosome via its interaction with CSN3For this purpose, we knocked-down PKD expression in mSos2-KO MEFs using specific siRNAs and then tested the level of Sos1 protein and various related cellular kinases by means of WB immunoassays using specific antibodies Fig. . Our pre6. A role of this region in intramolecular regulation of Sos GEF activity has been previously recognized19, but its overall functional relevance is not yet fully understood, and various regulatory questions concerning the domains within this region remain unclear.The proteins of the mammalian Sos family are composed by a linear sequence of functionally distinct modular domains. In this report, we focused on the N-terminal region of Sos1, encompassing the histone-like domain (HD), the Dbl-homology domain (DH), the pleckstrin domain (PH), and the helical linker (HL)43 are also able to interact with the COP9 signalosome.Looking for cellular proteins with putative binding affinity for the N-terminal region of Sos1, we carried out a yeast two-hybrid screening of a HeLa cDNA expression library and found strong positive interaction between CSN3 and the Sos1 histone-like domain (HD). This binding was confirmed in vitro by pull-down experiments with GST-fusion proteins and in vivo by means of co-immunoprecipitation assays in lysates of mouse and human cell lines, thus confirming the occurrence in eukaryotic cells of direct physical interaction between CSN3, a subunit of the COP9 signalosome, and the HD motif of Sos1. Further studies will be needed in future to pinpoint more accurately the exact region within the HD that is involved in this interaction and also to ascertain whether the highly homologous Sos2 proteins6. Previous structural studies showed that the HD is conformationally coupled to the DH-PH unit, therefore stabilizing the auto-inhibited conformation of Sos1, in which the DH-PH domain is also known to interfere with the allosteric binding motif for Ras22. Only upon ligand stimulation of upstream tyrosine kinase receptors (RTK), the second phase of Sos activation—(i) recruitment to the plasma membrane (involving PH binding to membrane phosphoinositides and electrostatic interaction between a positively charged patch of the HD and the negatively charged membrane) and (ii) disruption of the auto-inhibited conformation mediated by the DH-PH unit— can take place. Once in the membrane, the reoriented Sos molecules, displaying increased accessibility to both of its allosteric and catalytic Ras binding sites, can processively catalyze nucleotide exchange on multiple Ras molecules. Various mutations associated with Noonan syndrome, a disease caused by hyperactivated Ras signaling, are located in the HD and the PH domains45. Intramolecular interaction between the HD and the HL, located between PH and REM domains, may also contribute to maintain the inactive, auto-inhibited conformation of Sos since a mutation in the HL (R552G) increases the nucleotide dissociation rate of Ras and the mutated HL does not interact with the HD47. It will be interesting to determine in future whether or not any of the known Noonan HD mutations may affect the Sos1-CSN3 interaction in vivo.The HD contains two tandem histone folds and exerts negative control on the overall Ras-GEF activity of Sos1 under basal conditionsOur observations characterizing the interaction between CSN3 and the Sos1 HD suggest that this domain not only functions regulating Sos-GEF autoinhibition but is also involved in other functional roles, such as the control of Sos protein stability and homeostasis by modulating the degradation and intracellular levels of Sos1. On one side, our data showed that an in vivo interaction occurs between the COP9 signalosome and endogenous full-length Sos1 protein and that such an interaction is probably functionally significant as the interaction between them is higher upon RTK activation, probably due to disruption of the auto-inhibited conformation resulting from competition with, or uncoupling of, the basal HD/DH-PH interaction. On the other hand, our two-hybrid and co-immunoprecipitation experiments demonstrating physical interaction between the CSN3 and Sos1 support the notion of the participation of the UPS pathway in regulation of the homeostasis and intracellular levels of Sos1 in eukaryotic cells.34. CSN serves as a dominant regulator of cullin-RING ligase family of ubiquitin E3 complexes (CRLs)48 through removal of Nedd8 from cullins49. All CSN subunits are essential for full function of the complex and are produced in free-form before coordinated complex assembling26. Heterozygous deletion of CSN3 is the molecular cause of Smith–Magenis syndrome that results in reduction of the amount of the CSN protein complex50, according to the functional role of CSN3 for the CSN complex stabilization33.The COP9 signalosome(CSN) is a highly conserved, nine-subunit (CSN1–9) protein complex involved in the ubiquitin–proteasome system in eukaryotic organisms52. Specifically, PKD interacts with full-length CSN3 and modifies CSN727. In addition, CSN-associated PKD phosphorylates c-Jun and p53; CSN-mediated phosphorylation targets p53 for degradation by the UPS but stabilizes c-Jun, and inhibition of CSN-associated kinases triggers ubiquitin-conjugate formation and c- the case of Sos1, our data indicate that CSN3-associated PKD is able to phosphorylate Jun degradation27. Therefore, CSN acts as a platform recruiting protein kinases for regulating ubiquitin-conjugate formation by different E3 ubiquitin ligases27. In the case of Sos1, our data indicate that CSN3-associated PKD is equally able to phosphorylate the full-length Sos1 or a mutant lacking the HD domain. We also found that knockdown of all PKD forms or of CSN3, which destroys the CSN complex33, increases Sos1 degradation by the 26S-proteasome pathway. Future mechanistic studies will be needed to ascertain whether PKD mediates Sos1 stabilization through direct phosphorylation of the Sos1 protein at any of its conserved target sites for this kinase or through its interaction with CSN3 or other components of the COP9 signalosome. As several consensus PKD phosphorylation sites for Sos1  are also conserved in Sos2, these studies will also be relevant to analyze the possibility of dual control of the stability of both Sos1/2 isoforms via signalosome/PKD action.The CSN is associated with several protein kinases  able to modify substrates of the UPS, therefore affecting the stability of different cellular proteins27 could be a potential mechanism, the exact mechanistic details responsible for such a stabilization effect remain to be clarified by future studies. In any event, our data are consistent with the oncogenic role proposed for CSN53 and the CSN3 protein54 in different tumor types, and highlight the possibility that the CSN/PKD complex might be a potential molecular target for design of new therapeutic drugs.Taken together, our results strongly suggest that the CSN/PKD complex is involved in stabilization of Sos1 proteins. Although regulation of CSN-dependent deneddylation activity30 were maintained in DMEM supplemented with 10% fetal calf serum . Before immunoprecipitation, Sos1-KO and DKO cells were treated for 9 days with 0.3 µM tamoxifen before lysis. Cultures were tested routinely to exclude mycoplasma contamination . Transient transfections of HeLa and HEK293T cells were performed in p100 plates using Jet-Pei . For serum-starvation cells received DMEM containing 0.5% FBS 24 h after transfection and were then further incubated for 18 h. Cells were stimulated with 30% FCS for 10 min. All assays were done 48 h after transfection. Monoclonal mouse antibodies against CSN3, ubiquitin  and Sos1, and polyclonal rabbit antibodies against CSN7a, Sos1 (sc-256), Sos2 (sc-258), PKD1, and GST, were purchased from Santa Cruz Biotechnology . Monoclonal rabbit antibody anti-PKCα, clone M4 was purchased from Millipore; anti-HA and anti-AU5 monoclonal antibodies from Berkeley Antibody Company ; polyclonal rabbit antibodies against PKD2 and PKD3 from Cell Signaling; and polyclonal rabbit antibody against CSN3 was acquired from Abcam . Bound antibodies were detected in some experiments with secondary antibodies conjugated with IRDye680 or IRDye800 and analyzed with an Odyssey Imager system , or anti-mouse or anti-rabbit horseradish peroxidase  used as a secondary antibody were visualized using an Enhanced Chemiluminescence Detection Kit . 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside , 4-hydroxy-tamoxifen(TAM) (H7904), Cycloheximide (C1988) and MG132 (M7449) were from Sigma Co. . [γ32P]-ATP (370 MBq/ml) was from PerkinElmer, Inc. .NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% calf serum . Human HeLa cells, HEK293T cells, and mouse embryonic fibroblasts MEFs 55. pCEFL-KZ-AU5-hSos1 was cloned by amplifying hSos1 with specific primers providing Bcl-I and Sma-I sites at the 5ʹ and 3ʹ ends, respectively, and the amplified product was subcloned into Bgl-II and Eco-RV sites in pCEFL-KZ-AU5. To obtain HD-truncated mutant of hSos1 (pCEFL-KZ-AU5-hSos1-ΔHD), plasmid pCEFL-KZ-AU5-DH-PH was digested with Hind-III and Spe-I, and the AU5-DH-PH fragment was subcloned into Hind-III and Spe-I sites of pCEFL-KZ-AU5-hSos1. To previously generate pCEFL-KZ-AU5-DH-PH, the DH-PH domain of hSos1 (coding region 537–1653) was PCR-amplified from the pCEFL-KZ-HA-hSos1 plasmid using specific primers and providing sites BglII and NotI at the 5´ and 3´ ends, respectively. The amplified product was then subcloned into BglII and NotI sites of vector pCEFL-KZ-AU555. The HD and NDP domains of hSos1 were PCR-amplified using the specific primers  providing sites Eco-RI and Sal-I at the 5ʹ and 3ʹ ends, respectively. The amplified products were then subcloned into Eco-RI and Xho-I sites in pGEX-4T-1 (Amersham Biosciences). The DH-PH domain of hSos1 (coding region 537–1653) was PCR-amplified using specific primers and providing sites Eco-RI and Not-I at the 5ʹ and 3ʹ ends. The amplified products were then subcloned into Eco-RI and Not-I sites in pGEX-4T-1. pEFBOS-GFP fused to PKD1 kinase-dead  or constitutively active  were a gift from T. Iglesias56. The sequences of the oligonucleotides utilized are available upon request.The majority of hSos1 constructs were described previouslySaccharomyces cerevisiae EGY48 strain  was used as a host. Co-transformation of two-hybrid vectors into yeast were performed as described57. Protein–protein interaction in the two-hybrid system was monitored as described55.PCR-derived fragments encoding for the appropriate NDP, HD, DH-PH, DH, PH, and PRII domains  were subcloned into plasmid pEG202, encoding the LexA-binding domain. The full-coding sequence for CSN3 was cloned into plasmid pJG45, encoding an amino-terminal B42-activation domain. 55. Immune complexes were collected by centrifugation, washed five times with lysis buffer, resolved by SDS-PAGE and visualized by immunoblotting.Cells treated as indicated in each case were lysed in lysis buffer and nucleus-free supernatants were incubated with appropriate antibodies as described55. In all binding experiments, transfected mammalian cells were lysed in lysis buffer55 and 20 μg of the corresponding GST-fusion proteins, coupled to glutathione–sepharose beads, were used55.GST-fusion proteins were obtained and purified as describedIn knockdown experiments, siRNA duplexes (60 nM) were transfected twice in MEFs using Lipofectamine RNAiMAX Reagent (Invitrogen) at 24 h intervals. To knockdown CSN3 and PKD1/3, we used siGENOME SMART pool mouse Cops3 (M-047361-00), siGENOME SMART pool mouse Prkd1 (M-048415-01), siGENOME SMART pool mouse Prkd2 (M-040693), siGENOME SMART pool mouse Prkd3 (M-040692-00), and for siRNA control siGENOME control pool (D-001206-14-20), all purchased from Dharmacon.58 by incubating immunoprecipitated kinase (GFP-PKD-KD or GFP-PKD-CA) with eluted substrates or AU5-hSos1 or AU5-hSos1-ΔHD immunoprecipitates in the presence of ATP-gamma P32.In vitro kinase assays were performed as describedt test for unpaired observations. Western blots were analyzed using linear correlations between increasing amounts of protein and signal intensity.Data were analyzed using GraphPad Prism 7.0. Results expressed as mean ± SD of the indicated number of experiments. Statistical significance was evaluated using the


Composite materials usually possess a severe deformation incompatibility between the soft and hard phases. Here, we show how this incompatibility problem is overcome by a novel composite design. A gradient nanolayer-structured Cu-Zr material has been synthesized by magnetron sputtering and tested by micropillar compression. The interface spacing between the alternating Cu and Zr nanolayers increases gradually by one order of magnitude from 10 nm at the surface to 100 nm in the centre. The interface spacing gradient creates a mechanical gradient in the depth direction, which generates a deformation gradient during loading that accumulates a substantial amount of geometrically necessary dislocations. These dislocations render the component layers of originally high mechanical contrast compatible. As a result, we revealed a synergetic mechanical response in the material, which is characterized by fully compatible deformation between the constituent Cu and Zr nanolayers with different thicknesses, resulting in a maximum uniform layer strain of up to 60% in the composite. The deformed pillars have a smooth surface, validating the absence of deformation incompatibility between the layers. The joint deformation response is discussed in terms of a micromechanical finite element simulation. A key goal in composite design is to utilize and combine the properties of the constituents, e.g., the deformability and lightness of the soft component and the high stiffness and strength of the hard component, to achieve the best advantages. For example, this combination is realized in metal matrix composites4 and carbon nanotube-reinforced composites7. However, composites are prone to severe deformation incompatibility between constituent phases that naturally possess a large mechanical mismatch. For example, the deformation incompatibility in metal matrix composites leads to pull-out of the carbon fibres9, the formation of cavities10, or premature fracture of the particulates or interfaces12. Another typical example is the shear instability observed in multilayered/phased nanostructures, such as shear cracks, shear bands or extrusion of soft materials24. These incompatibilities can drastically reduce the mechanical performance and service life of composite materials. Despite efforts to alleviate the incompatibility problem26, there is still no general strategy for eliminating deformation incompatibility in composite materials. We therefore designed a gradient nanolayer-structured composite material by the alternating stacking of soft Cu and hard Zr nanolayers with a gradient distribution in the layer thickness from the surface to the centre. The new design creates an interface spacing gradient structure. This design was inspired by the existing natural and artificial gradient structures, which show extraordinary deformability and compatibility31.Composite materials play a central role in the development of novel engineering solutions, particularly in the aerospace and automobile sectors28, such as the fibre density gradients in bamboo32, the alignment gradients in pangolin armour33 and the gradients in the chitin-calcite plywood structure of the exoskeleton of arthropods34. These gradients render biological materials resistant against harsh mechanical loads such as those imposed by weather or predators. Recently, manmade gradient structures have also been shown to provide improved strength-ductility properties47, lower friction coefficients48, better fatigue resistance51, and exceptional stretchability52. The underlying artificial gradient structures that were utilized in these studies mainly included grain size gradients spanning over four orders of magnitude53 or nanotwin gradients in twinning induced plasticity steel54. These gradient approaches have proven to be a successful strategy for enhancing the mechanical properties through defect gradients without requiring alloy modifications55, i.e., they were applied to pure metals or homogeneous solid solution alloys. Here, we extend this idea to a multiphase composite material in order to take the first step towards solving the general problem of the deformation incompatibility encountered in nanolayered composites by utilizing structural gradients. We used micropillar compression to test the deformation compatibility in the gradient architecture under nearly uniaxial stress conditions during deformation due to the fact that pillars have no confining material and, hence, reveal any deformation mismatch at the free surfaces upon mechanical loading.Natural gradient structures are ubiquitous26. We used geometrically necessary dislocations (GNDs) to achieve this goal. According to the classical work of Ashby56, GNDs accommodate a gradient of deformation in non-homogeneous materials and thus enable compatible deformation. The above conclusion has been validated by many scientists61. The aim of the nanolayer gradient design is to form a mechanical gradient in terms of strength and strain hardening, and in turn, non-uniform deformation in a gradient shape along the depth direction of the architecture. The ultimate goal is to generate sufficiently high populations of GNDs through gradient deformation to achieve strain compatibility between all the Cu and Zr layers with various thicknesses. Indeed, the new nanolayer gradient architecture enables large synergetic deformation (up to 60% uniform layer strain in the composite) between the layers with thicknesses varying from 10 nm to 100 nm. After loading, the pillars show a regular and smooth surface, indicating a considerably improved strain compatibility between the layers of soft Cu and hard Zr phases with various layer thicknesses. We analysed the results and the boundary conditions using a micromechanical finite element (FE) simulation, which models the individual layers and their co-deformation in terms of an elastic-plastic constitutive description  Cu/Zr composites were synthesized by magnetron sputtering (see Methods). The individual Cu or Zr layer thickness is designed to vary from 10 nm at the bottom to 100 nm in the centre and then from 100 nm back to 10 nm in the top half, thus producing an overall symmetric through-thickness layer distribution. Two gradient nanolayer samples, GNL1 and GNL3, were prepared, in which multiple Zr-Cu bilayers with constituent thicknesses of 10 nm, 20 nm and 30 nm were included in one half of the sample, and the numerals ‘1’ or ‘3’ indicate the number of the above bilayers of each thickness. The nominal total thicknesses of the two gradient samples are 1.12 μm and 1.6 μm, respectively. Figure 62, Cu/Cr63 and Cu/amorphous CuZr66 systems, were not observed. The corresponding EDS maps of Cu and Zr also prove that no internal mixing, i.e., no severe cross-layer deformation, occurred  accommodates the gradient deformation  silicon wafer through magnetron sputtering (Bestec PVD cluster). A 100 nm-thick Cu seed layer was deposited directly on the Si wafer before preparing the layered structure. The sputter targets are a Zr target of 99.9% purity and a Cu target of 99.99% purity. Both targets have a diameter of 76.2 mm and a thickness of 3 mm. Radio frequency sputtering is used to deposit Cu, whereas direct current sputtering is used to deposit Zr. The sputtering parameters were set as follows: the base pressure was less than 10D) and inner (d) diameters with correspondingly reduced beam currents. The adopted diameters (D and d), cutting depth (z) and beam currents for all the patterns are listed in Supplementary Table The pillar was prepared by focus ion beam (FIB) milling (FEI nanolab 600 and 600i). The targeted diameter is 600 nm. In order to minimize the taper of the prepared pillar, we selected an appropriate set of annular patterns with decreasing outer (Micropillar compression tests were carried out by using a 10-μm-diameter diamond flat punch (Nanomechanics InSEM-III) under a constant loading rate of 0.01 mN/s. The engineering stress-strain response was then calculated using the load-displacement records, taking the specimens’ cross-section at 20% of the pillar height from the top as a reference to translate the forces into stresses  and scanning electron microscopy . TEM and STEM were used to characterize the deformation compatibility of the pillars. TEM images, including diffraction contrast bright field and dark field images, were taken on a Philips CM20 microscope operated at 200 kV. STEM micrographs were obtained on a FEI Titan Themis microscope operated at 300 kV, using an aberration-corrected probe with ~0.1 nA current and a convergence semi-angle of 24 mrad, and the high-angle annular dark field (HAADF) detector with collection semi-angles of 73~352 mrad. We also collected the energy dispersive X-ray spectroscopy (EDS) spectra by a windowless, four quadrant silicon-drift-detectors covering a solid angle of 0.7 sr.A MicroMaterials Platform 3 nanoindenter with a diamond Berkovich indenter was used to measure the elastic modulus of the two gradient samples, i.e. GNL1 and GNL3. A total of nine indents were performed for each sample. The diamond area function of the tip was calibrated prior to indentation on a fused silica standard. A Poisson’s ratio of 0.33, i.e. the same as that of copper and close to that of Zr (0.34), was assumed for the conversion of reduced modulus to Young’s modulus.Supplementary material


KRAS突变、PTEN缺失和MET扩增；然而现在研究的更加深入的是获得性耐药，它的机制主要有：EGFR的二次突变、下游信号通路的激活、表型转变和EGFR突变之外的遗传学改变。在众多EGFR-TKI耐药机制中，肝细胞生长因子（hepatocyte growth factor, HGF）/上皮间质转化因子（mesenchymal-epithelial transition factor, MET）信号通路的异常激活在原发性耐药和继发性耐药中都发挥了重要作用，也是近年来研究的热点，研究人员也开发出了许多针对该通路的靶向药物。表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-TKI）现已成为治疗非小细胞肺癌（non-small cell lung cancer, NSCLC）的常规手段之一，但在临床上这类药物并不适用于所有NSCLC患者，大部分敏感突变的患者在经过大约12个月的初始治疗后都产生了获得性耐药，产生耐药的机制很复杂，其中原发性耐药的机制主要包括EGFR耐药突变、T790突变、11.1，在体内，生理条件下HGF是一种具有多种功能的生物因子，它参与血管生成、组织再生及免疫活性的调节。HGF也叫做散射因子，是纤维蛋白溶酶原家族成员，HGF在体内以单链前体被合成，在蛋白水解酶作用下水解成有活性的双链分子，成熟的HGF是由α链和β链经二硫键链接而成的异二聚体，具有活化MET的功能1.2MET是一个原癌基因，包含以下结构区：Sema区（Sema domain）、四个IPT区（immunuoglobulin-plexin-transcription domain）、PSI区（Plexins, semaphorins and integrins domain）、JM区（Juxtamembrane domain）、TK区（tyrosine kinase catalytic domain）和TM区（transmembrane domain）。Sema区为配体结合区，JM区有负调节激酶的活性，TK区则包含多个酪氨酸磷酸化位点并具有启动酪氨酸激酶活性的作用。，是一种跨膜酪氨酸激酶受体，与配体HGF结合后触发受体二聚化，进一步启动下游信号通路。HGFR可表达于人体许多器官或者组织的细胞内，包括肝脏、胰脏、前列腺、肾、肌肉和骨髓等，正常生理情况下它的活化能激活很多不同的细胞内信号通路，从而影响细胞的增殖、运动、移行、浸润等功能。HGFR在多种恶性肿瘤中存在调节异常，在肿瘤细胞的增殖、细胞扩散、血管形成、细胞粘附、细胞侵袭、细胞运动和抗凋亡中发挥着重要作用。肝细胞生长因子受体（hepatocyte growth factor receptor, HGFR）是MET编码的蛋白质产物2Src基因家族同源区-2（Src homology 2 domain-containing protein, Shc）、PI3K、磷脂酶C（phospholipase C, PLC）、酪氨酸磷酸酶SHP2和信号转导转录激活子3（signal transducer and activator of transcription 3, STAT3），通过这些信号蛋白进一步激活下游几个重要的通路：PI3K-Akt信号通路、Ras-MAPK信号通路和STAT3通路。HGFR的Sema区与HGF结合后，TK区便发生二聚化和磷酸化，HGFR的羧基末端会募集一些衔接蛋白，如生长因子结合蛋白2（growth factor receptor-bound protein 2, Grb2）、Grb2连接蛋白1（Grb2-associated binding protein, GAB1）和非受体酪氨酸激酶Src，之后Grb2和GAB1可以吸引更多的信号蛋白，包括, 13。HGF/MET也被认为与血管生成和内皮细胞功能调控有关，在多个模型中，HGF/MET信号激活可以诱导包括血管内皮生成因子（vascular endothelial growth factor, VEGF）在内的血管生成因子，并且与VEGF2协同参与了肿瘤的早期形成, 15。HGF/MET信号途径主导的这些功能的一个潜在机制是短暂的上皮间质转化（epithelial-mesenchymal transition, EMT），其特征是上皮分化、细胞扩散和迁移以及细胞外基质降解的缺失，在恶性肿瘤中，对这些进程严格调控能力的丧失导致了侵袭和转移发生, 17。生理状态下HGF/MET信号通路在胚胎和成人机体内都有表达，在胚胎发育过程中，HGF/MET信号通路在有丝分裂、形态生成等过程中扮演重要角色，在成人体内，该信号途径则在组织损伤后的修复和再生中发挥作用3MET突变。其中发生率最高的是HGF和HGFR过表达，Tachibana等利用FISH和IHC检测了106例肺腺癌患者的HGF和HGFR的表达率分别为17.9%和28.3%，并且在浸润性腺癌中的表达明显强于非浸润性腺癌。最新的研究发现在ALK（+）的NSCLC患者中，HGFR的表达水平要明显高于ALK（-）患者。HGF/MET信号通路异常激活包括HGF和HGFR过表达、MET扩增和MET基因拷贝数的作用越来越受到人们重视，Park等检测了NSCLC患者组织中EGFR和MET的相对拷贝数，发现EGFR和MET在非小细胞肺癌的发生和发展中可能存在相互或者协同作用，并能对非小细胞肺癌患者进行预后评估。此外，有研究者分析了213例NSCLC患者的病理切片，发现MET基因拷贝数增加是术后NSCLC患者预后不良的独立因素。近年，MET突变可以发生在Sema区、JM区和TK区，Sema区发生的错义突变与MET二聚化有关，且易发生于鳞癌和吸烟患者，JM区的基因突变则与肿瘤发生有关, 23。MET编码序列点突变的发生率很低，但在许多实体瘤中都有报道，在NSCLC中，44.1发现在EGFR-TKI原发性耐药的肺腺癌细胞系中，HGF呈现出了异常的高表达，这提示HGF吉非替尼原发性耐药中扮演着重要角色，HGF表达增加会过度激活MET介导的PI3K-Akt信号通路，降低EGFR-TKI对这种信号级联反应的抑制，与获得性耐药不同，原发性耐药主要是由HGF刺激MET通过GAB1而不是ERBB3的作用激活下游信号通路。HGF/MET信号通路在NSCLC原发性耐药和继发性耐药中都发挥了重要作用。Yano等通过建立的吉非替尼耐药细胞系的MET活化模型，发现如果细胞存在大量MET活化，那么EGFR-TKI对癌细胞的作用将大大受限，即便是敏感突变的细胞也是如此，这也表明MET活化与EGFR-TKI的原发性耐药有着密切关联。最新的一项研究发现HGF的存在可导致EGFR酪氨酸激酶的失活，从而引起EGFR-TKI的原发性耐药，同时诱导EGFR与其他的肿瘤相关蛋白相互作用，结论认为应用EGFR-TKI治疗上皮来源肿瘤之前采用HGF/MET抑制剂预处理可防止耐药的发生。Benedettini等4.2MET扩增在EGFR-TKI耐药原因中分别占61%、52%和9%，耐药组织中HGF表达水平要明显高于EGFR-TKI敏感组织（P < 0.001），一半的耐药肿瘤样本同时存在HGF过表达、T790M突变和MET扩增。在体外，通过同时抑制HGF高表达、T790M突变和MET扩增这三种耐药机制，研究者发现联合EGFR-TKI（WZ4002）和MET-TKI（E7050）能够有效对抗细胞株对厄洛替尼的耐药性。在没有EGFR T790M和MET扩增的配对样本中，治疗后出现耐药组的HGF表达明显高于治疗前的样本（P=0.025），这说明HGF是促进药物耐药的一个独立因素，与MET原癌基因扩增是相互独立的；对耐药患者血液中HGF的检测也得到了一致的结论。在亚洲人群中，HGF高表达、T790M突变、表明HGF可诱导敏感肺癌细胞PC-9和H292对吉非替尼耐药，并且HGF刺激MET磷酸化可能是敏感肺癌细胞对吉非替尼耐药的重要机制。研究还发现在EGFR-TKI环境下，HGF可以通过促进MET扩增增强细胞的耐药性。HGF过表达还与NSCLC患者的不良预后有关，数据显示HGF过表达和低表达患者的5年生存率分别为22.2%和75.0%（P=0.259）, 34。最新研究MET扩增率约为5%-22%。Engelman等建立了对吉非替尼产生获得性耐药的肺癌细胞株，并对其进行干预，发现MET原癌基因的扩增可以通过激活依赖EGFR的ERBB3磷酸化和下游PI3K/AKT通路避开EGFR-TKI的靶点，从而导致NSCLC对EGFR-TKI产生耐药。数据显示，在对吉非替尼或厄洛替尼耐药的肺腺癌患者中，有21%的病例出现MET扩增，而没有用吉非替尼和厄洛替尼患者的MET扩增率只有3%，这提示MET扩增与EGFR-TKI耐药有关。虽然MET扩增和EGFR T790M突变可以同时出现，但T790M和MET基因拷贝数呈负相关，表明两者在获得性耐药机制中是互补的关系。在出现EGFR-TKI获得性耐药的NSCLC患者中，5。还有一种通过小分子MET抑制剂可以在酪氨酸激酶区水平抑制该通路。近来，有一大批针对MET酪氨酸激酶区的小分子药物进入临床试验，已经进入临床观察的MET-TKI：Tivantinib（ARQ 197)、Foretinib（XL880）和Cabozantinib（XL184）。自从发现HGF/MET信号通路是导致EGFR-TKI耐药的一个重要因素后，人们便不断寻找克服的方法，研发针对HGF/MET信号通路的靶向药物也成为目前靶向治疗研究的一个热点，主要是在以下两个传导水平抑制该通路：第一种是通过抗HGF或者HGFR在配体/受体水平阻断该信号通路，已进入Ⅱ期临床试验的代表药物包括：①抗HGF的单抗：AMG-102（rilotumumab）和AV-299（ficlatuzumab）；②抗HGFR单抗：MetMab（onartuzumab）已经进入了Ⅲ期临床试验，并得到了可喜的结果虽然针对HGF/MET信号通路的靶向药物层出不穷，但现在学者们普遍认为针对不同耐药机制的联合靶向治疗是克服耐药的最好方法。6总之，HGF/MET信号通路在人体生理功能中不可或缺，其异常激活在肿瘤发生、发展和靶向治疗耐药方面也发挥着重要作用。随着靶向治疗耐药机制研究的不断深入，很多新的靶向药物已进入临床试验，相信不久的将来，NSCLC患者会有更多更好的靶向治疗选择。


Radiometric calibration for imaging sensors is a crucial procedure to ensure imagery quality. One of the challenges in relative radiometric calibration is to correct detector-level artifacts due to the fluctuation in discrepant responses  and electronic instability . In this paper, the integration of the empirical mode decomposition (EMD) with Hilbert–Huang transform (HHT) in relative radiometric calibration was explored for a new sensor, FS-5 RSI (remote sensing instrument onboard the FORMOSAT-5 satellite). The key intrinsic mode functions (IMFs) analyzed by HHT were examined with the pre-flight datasets of the FS-5 RSI in temporal and spatial variations. The results show that the EMD–HHT method can stabilize and improve the radiometric quality of the FS-5 imagery as well as boost its application ability to a new level. It is noticed that the IMFs of the spatial variation would be disturbed by the instability of the temporal variation. The relative response discrepancies among detector chips can be well calibrated after considering the temporal effect. Taking a test imagery dataset of gain setting G2 as an example, the standard deviation (STD) of the discrepancy in the digital number after calibration was dramatically scaled down compared to the original ones . The good performance of pre-flight imagery indicates that the EMD–HHT approach could be highly practical to the on-orbit relative radiometric calibration of the FS-5 RSI sensor and is applicable to other optical sensors. To our knowledge, the proposed EMD–HHT approach is used for the first time to explore relative radiometric calibration for optical sensors. Sensors of modern digital imagers onboard satellites must be assured of accuracy by pre-flight radiometric calibration. This task is relatively easy in the space environment simulation laboratory prior to a satellite’s launch. However, after launch, sensors are apt to experience degradation over time which may occur in optical, electrical or supportive components during launch; or due to satellite orbital drifts and perturbations, cosmic ray bombardment, etc. As illustrated in The FS-5 project is the first space program for which the National Space Organization (NSPO) has taken full responsibility for the design, development, and system integration. Its mission is to promote space science experiments and research, to enhance Taiwan’s self-reliant space technology capabilities, and to continue to serve the users of FS-2’s global imagery services. The NSPO developed the key components of the remote-sensing instrument (RSI) sensor and spacecraft bus through the integration of resources from domestic partners ′ is the grayscale value after subtracting the contribution of the dark current C0. Therefore:C(R)]′ = R′. However, the sensor’s response is non-uniform, as seen from the test imagery dataset of the FS-5 , which was developed by Huang et al. in 1998, is quite suitable for analyzing and processing non-steady-state and non-linear signals. Thus, it can be applied to correct the test imagery dataset of FS-5 under different electronic gain settings either in digital mode or flying mode ,6.The combination of the Hilbert spectral analysis (HSA) and empirical mode decomposition (EMD) is designated as HHT by NASA . HHT is superior to Fourier transform or wavelet transform in terms of providing better time-domain and frequency-domain resolution for the input data set. The key part of HHT is EMD, through which any complicated dataset can be decomposed into multiple intrinsic mode functions (IMFs). Each IMF represents a narrow band frequency-amplitude modulation that is often related to a specific physical process. The EMD method is adaptive, data-driven, and highly efficient. It is applicable to non-linear and non-stationary processes since the decomposition is based on the data’s local characteristics ,27. EMD x(t) are decomposed in terms of IMFs, jc:nr is the data residual of x(t) after the IMFs have been extracted n times. The EMD is implemented through a sifting process that uses only local extrema. For any dataset, x(t) = jr−1, the procedure involves the following three steps: (1) take all of the local extrema (both maxima and minima), connect them with a cubic spline as the upper (lower) envelope, and calculate the corresponding local mean; (2) extract the first component H by subtracting local mean from the data x(t); and (3) treat H as the new data and repeat steps 1 and 2 until the envelopes are symmetric at about zero to a certain threshold. The final H is designated as jc. When the residue nr becomes a monotonic function or when no more IMFs can be extracted from the above procedure, the sifting process is finished. The secular trend of a time series is then obtained after all of the oscillatory components (riding waves) are removed by applying EMD. Depending on the total number of data points N, the derived number of IMF components are approximately as log2N. The specific steps of IMF shifting are illustrated in EMD is the empirical and adaptive algorithm for the accurate expression of a time series in the time-frequency-energy domain. In EMD, the data xC is the relative grayscale coefficient, which is defined as the ratio of the calculated grayscale value over the baseline grayscale value; Note that a horizontal line over the denominator equation denotes the average/mean grayscale value which served as the baseline (reference) grayscale value. x is the number of accumulative deducted IMFs; x is determined by the lowest STD value of each band shown in the calibration result from x was chosen based on the lowest STD, which implies the lowest level of uneven responses among the detector chips of sensor array. The relative response coefficient of each detector chip, ρ(p) in Equation (3b), is then obtained by the radiometric conversion Equation (1c). Procedures of the relative radiometric calibration proposed in this study are illustrated in Based on the EMD algorithm with the inputs transformed by HHT (referred to as EMD–HHT), a set of IMFs can be generated, taking that the mean grayscale value of the FS-5 RSI test imagery is the baseline (reference) grayscale value. Then, by deducting various accumulative IMFs from the original grayscale value, i.e., the sum of accumulative IMFs subtracted from the original grayscale value, we derive the relative grayscale coefficient, as given in the following equation:i-th detector chip of the sensor obtained by the EMD method proposed in this study; i-th detector chip of the sensor obtained by the method of the NSPO. The NSPO method includes a normalizing procedure based on the uniform radiance scene and zero radiance (darkness) scene, which is an on-ground/pre-flight radiometric calibration approach.The relative response coefficient of the sensor’s detector chips obtained in this study is compared with that calculated by the NSPO, in which the normalization method is applied ,12,18. TPrevious methods of relative radiometric calibration are generally focused on the unevenness of a sensor’s detector chips, known as the spatial variation of the image. Taking the test imagery of FS-5 as an example, ρ(p). For instance, after calibration in the spatial variation, IMF-filtered images of the flying mode and digital mode can be obtained as shown in Relative radiometric calibrations for the spatial variation tackle the inhomogeneity of the individual detector chips of the sensor array. According to Equation (3b) in To evaluate whether the EMD–HHT method is a good approach for relative radiometric calibration, the performance of the EMD method calibration for each IMF-filtered imagery is assessed based on the STD. The FS-5 RSI optical imager is equipped with a PAN band of 12,000 detectors (pixels) and multispectral (MS) bands of 6000 detectors (pixels). The STD is calculated for each cross-track line ) after the temporal density calibration is generally lower than that without the temporal density calibration; that is, the calibration performance is even more effective after the temporal density calibration procedure.In addition, comparing the calibration result between digital mode and flying mode test imagery, it can be seen that in order to achieve the lowest STD value, the accumulative number of IMFs for filtering  and (3b), we derived the relative response coefficients for all gain settings and bands, then compared and verified with the relative response coefficients provided by the NSPO, as shown in To display the relative difference between the spatial density calibration and the combined temporal and spatial density calibration, we calculate the relative response coefficients of the sensor’s detector chips using two different calibration results. The relative response coefficient of the sensor’s detector chips of flying mode and digital mode test imagery datasets can be obtained by the calculation of Equations (3a) and (3b). Field-test imagery after calibration by applying the relative response coefficient are shown in The application of the relative response coefficient to pre-flight imagery can be a simulation of the on-orbit calibration procedure, as demonstrated by the results above. Taking a uniform surface as the calibration region, such as ocean or desert areas, the EMD–HHT method can facilitate the periodical maintenance of relative response coefficients for FS-5 RSI.(1)The striping and banding noises of FORMOSAT-5 RSI imagery may be caused by inconsistencies between detector chips and electronic instability in the spatial and temporal variations.(2)The calibration of the temporal variation to stabilize essential IMFs of the spatial variation is suggested, although the temporal variation of each detector is relatively small.(3)Testing results show that the EMD–HHT method of relative radiometric calibration proposed in this study is able to eliminate the striping and banding of the test imagery dataset caused by the non-uniformity of sensors. The overall improvement rate is over 95% for all spectral bands and gain settings. The temporal relative radiometric calibration subsequently reduces the accumulative numbers of IMFs required for filtering as well as the post-calibration mean STD, indicating the significance of and need for relative radiometric calibration in the temporal variation.(4)In comparing the relative response coefficients of images from flying mode sensors (examined with a standard light source only), the mean error between results of the EMD–HHT approach and that provided by the NSPO was 0.006%. This shows that EMD–HHT for the relative radiometric calibration of FS-5 RSI sensors is highly effective and its transferability to other sensors is promising as well.(5)For digital mode sensors, relative response coefficients are derived from the images viewed with a uniform light source, and then applied to the field images in order to filter out the striping and banding noises. The results, as demonstrated in The FS-5 RSI is the successor to the FS-2 RSI, providing high spatial-resolution imagery at the global scale for environmental resources and disaster-monitoring services. The quality of FS-5 RSI imagery is thus essential to the capability of its further applications. Because the pre-processing system for the testing of both geometric and radiometric data is not yet ready, the mission of FS-5 RSI is still pending at this moment. The approach of relative radiometric calibration proposed in this study could be a great benefit to the radiometric pre-processing procedure for FS-5 RSI on-orbital imagery. The results in comparison with those provided by the NSPO reveal that the proposed approach, the EMD–HHT method, can be expected to offer near real-time calibration for the maintenance of the relative response coefficient after FS-5 RSI operation. According to the EMD–HHT method conducted on FS-5 RSI images in the digital mode and flying mode, the following conclusions have been drawn:On a final note, the discrepant response between detectors in the spatial variation is more straightforward than the instability caused by the electronic system in the temporal variation. That is, the relative discrepancy in the spatial variation is basically fixed while the electronic instability in the temporal variation may be varied. The identification of the periodic cycle of fluctuation in the electronic system is significant to the relative calibration in the temporal variation. Therefore, exploring the fluctuating cycle of the electronic system with on-orbit FS-5 RSI images over uniform surface areas for the application of the EMD–HHT method for relative radiometric calibration is the next step in this field of research.


Due to their capacity to process different proteins of the extracellular matrix (ECM), matrix metalloproteinases (MMPs) were initially described as a family of secreted proteases, functioning as main ECM regulators. However, through proteolytic processing of various biomolecules, MMPs also modulate intra- and extracellular pathways and networks. Thereby, they are functionally implicated in the regulation of multiple physiological and pathological processes. Consequently, MMP activity is tightly regulated through a combination of epigenetic, transcriptional, and post-transcriptional control of gene expression, proteolytic activation, post-translational modifications (PTMs), and extracellular inhibition. In addition, MMPs, their substrates and ECM binding partners are frequently modified by PTMs, which suggests an important role of PTMs in modulating the pleiotropic activities of these proteases. This review summarizes the recent progress towards understanding the role of PTMs  on the activity of several members of the MMP family. Matrix metalloproteinases (MMPs) comprise a family of 23 distinct secreted or membrane-anchored endopeptidases in humans that belong to the metzincin superfamily of metalloproteases. MMPs were initially described as regulators of the extracellular matrix due to their capacity to degrade ECM proteins like collagen, gelatin, laminin, aggrecan, fibronectin, elastin, and proteoglycans. Based on structure and substrate specificity, the MMPs are divided into collagenases , gelatinases , stromelysins , matrilysins , membrane-type MMPs , and other non-classified family members . MMPs share a multidomain structure consisting of signal peptide, zymogenic pro-peptide domain, a catalytic domain, a variable linker ‘hinge’ region and a hemopexin domain. Some MMPs show variation in the domain arrangements, e.g., MMP7 and MMP26 are lacking the linker and hemopexin domain, MMP23 has a unique cysteine-rich and immunoglobulin-like instead of a hemopexin domain, and the two gelatinases MMP2 and MMP9 have additional fibronectin type-II-related domains. Membrane-type MMPs are anchored to the membrane either via glycosylphosphatidylinositol (GPI) or with help of a transmembrane domain . AdditioMMPs are primarily extracellular proteases, supporting the initial concept that they are generally associated with degradation and regulation of the ECM, thereby influencing many fundamental cellular events involving ECM remodeling ,3,4,5. M2-macroglobulin . Und. Und1]. N- and O-linked glycosylation [N-linked glycosylation is a glycosidic linkage of glycans to the side chains of asparagine (Asn) residues. It is initiated by a single oligosaccharyltransferase complex with a transfer of N-acetylglucosamine (GlcNAc) phosphate at the consensus Asn-Xaa-Ser/Thr sequence  on the cytosolic side of the endoplasmic reticulum (ER) and extensively modified further in the lumen of the ER and Golgi prior to sorting to secretory vesicles [O-linked glycosylation is a glycosidic linkage of glycans to the side chains of serine/threonine (Ser/Thr) or tyrosine (Tyr) residues. O-linked glycosylation shows a higher diversity than N-glycans, resulting from the complex synthesis of N-acetylgalactosamine -type O-linked glycans in the Golgi, which can be initiated by up to 20 different polypeptide GalNAc-transferase isoforms that may compete for the same glycosylation site and the same glycan. Upon transfer of a GalNAc moiety from a donor substrate uridine diphosphate (UDP)-GalNAc to Ser/Thr residues, the O-linked glycan is branched out by different core-forming enzymes and further extended by fucosylation and sialylation to the final O-linked glycan structure [N- and O-linked glycosylation are present across the MMP family [Glycosylation describes the enzymatic reaction that links saccharides to lipids, other saccharides or proteins and represents the most abundant and diverse PTM. The most common forms of protein glycosylation are the sylation ,38. N-livesicles ,39,40,41tructure ,43,44,45tructure ,48,49,50tructure ,52. The N-linked glycosylation sites, Asn38 and Asn120 (asparagine residues 38 and 120), in the pro-domain and the catalytic domain, respectively [N-linked glycans have been described as core-fucosylated biantennary structures, partially sialylated with variable fucosylation branches [N-linked glycosylations are distinct, primarily due to their location within the protease. Since N-linked glycosylation is generally required for protein secretion, initially a potential role of these two glycans was related to the secretion of MMP9 [120 reduced the efficiency of its secretion by increasing the interaction between MMP9 and calreticulin (a protein that prevents misfolded proteins from entering the secretory pathway), thereby inducing MMP9 retention in the ER. The glycosylation at Asn38 does not influence the secretion of MMP9, but Asn38-glycosylation-deficient MMP9 shows strong amino-acid dependency towards interaction with calreticulin, likely affecting MMP9 secretion in an N-glycosylation-independent manner [N-glycosylation at Asn38 was commonly associated with the activation of proMMP9, yet the process of MMP9 activation occurs independently of the glycosylation at Asn38. Recent molecular dynamics simulations suggest that the glycosylation at Asn38 is indirectly involved in the activation of proMMP9 by inducing conformational changes within the pro-domain, enabling MMP3 to access the two cleavage sites for proteolytic activation [38 and galectin 3 decrease the proteolytic activation of MMP9, whereas interactions with galectin 8 enhance the MMP3-mediated processing, suggesting that the presence of N-glycosylation is important for a fine-tuned regulation of MMP9 activity [O-linked glycans (denominated OG domain) [O-linked glycans comprise a heterogenic mixture, varying from core-1  to core-2 GalNAc) structures and further elongated to larger glycans [O-linked glycans increases the domain flexibility of MMP9, allowing the protease to adopt multiple enzyme conformations and facilitating individual movements of the catalytic and the hemopexin domain. This influences the recognition, binding, and processing of substrates, cell receptors, and endogenous inhibitors [MMP9 is the most extensively glycosylated MMP. This protease contains two ectively . These Nbranches . Despite of MMP9 . Indeed,t manner . Additiotivation . Moreoveactivity ,59. Furt domain) . Based o glycans . The prehibitors ,63. A sthibitors . Consequhibitors . Howeverhibitors . An indehibitors . Furtherhibitors .O-linked glycosylation sites  identified in the linker region. The presence of these O-linked glycans does not affect zymogen activation, collagenase activity or the autolytic processing of MMP14. However, the O-linked glycosylation is important for the formation of a stable complex between MMP14, TIMP2, and proMMP2, which is required for subsequent cell-surface activation of MMP2. Being unable to recruit TIMP2, which connects the catalytic domain of MMP14 and the hemopexin domain of proMMP2, the glycan-deficient MMP14 cannot present a stable trimeric complex on the cell surface and thereby activate MMP2 [Similar to MMP9, the transmembrane collagenase MMP14 (MT1-MMP) contains ate MMP2 ,66,67. Iate MMP2 .N-glycosylation at Asn120 has been experimentally confirmed. Comparison between the glycosylated and non-glycosylated MMP1 showed no significant differences in activity, substrate specificity or inhibitory profiles of the two proteoforms. However, the presence of specific glycan motifs  can initiate MMP1 selectin-mediated binding to the surface of activated cells through a selectin/glycan interface and therefore may have a profound effect on cell migration [MMP1 has two potential glycosylation sites in the active site, but only igration ,69.N-linked glycosylation sites, Asn573 and Asn642, in the hemopexin domain [N-linked oligosaccharides remains unclear. However, there are strong implications regarding their involvement in the regulation of MMP2, since the hemopexin domain is involved in MMP2 activation/inhibition, localization of its catalytic activity, and induction of cell signaling upon interaction of MMP2 with cell-surface receptors [MMP2 has two potential n domain ,71. The eceptors .N-linked glycosylation, MMP3 also has two potential N-linked glycosylation sites, Asn120 in the catalytic domain and Asn398 in the hemopexin domain. However, only a small portion of MMP3 (~20%) is glycosylated, and no correlation has been established between these glycans and the function of the protease [Based on the consensus sequence for protease .117 and Asn152) in the catalytic domain. The N-linked glycosylation site at Asn117 has been experimentally verified, but the function of this N-linked glycan has not been determined since no differences were observed between glycosylated and non-glycosylated recombinant MMP13 [MMP13 was shown to be potentially glycosylated at two asparagine residues , a glycosylphosphatidylinositol-anchored matrix metalloproteinase, has two sulfides ,77,78.Protein phosphorylation is one of the most important PTMs for regulation of biological processes . This dyAs many secreted and extracellular proteins, the MMPs are phosphorylated. Ser-, Thr-, and Tyr phosphorylation sites are identified across the different structural domains of the MMPs. Yet, the biological significance of these phosphate groups is overall poorly characterized. Among the MMPs, MMP2 and membrane-type MMP14 metalloproteinase are the ones with most extensively studied phosphorylation-dependent effects.MMP2 contains 29 potential phosphorylation sites distributed across the pro-peptide domain, collagen-binding domain, collagenase-like domain-1 and -2, and hemopexin domain. However, only five of the predicted phosphorylation sites  have been confirmed by mass spectrometry (MS). The phosphorylation of MMP2 noticeably diminishes its proteolytic activity, while dephosphorylation increases MMP2 activity. This is possibly due to the conformational changes observed in the secondary structure of dephosphorylated MMP2, with α-helices 50% longer and β-strands 17% shorter than phosphorylated MMP2. However, it is still unclear if protease activity is directly influenced by these conformational changes ,84.567 and Tyr573, which have a significant influence on MMP14-induced cellular invasion and migration. A study by Williams et al. in fibrosarcoma cells showed that substitution of the residue Thr567 by alanine increased retention at the cell surface and reduced internalization of the protease significantly compromising invasion and migration. Contrarily, mimicking protease phosphorylation by substitution of Thr567 by glutamic acid reduced retention of MMP14 at the cell surface, increased efficiency of internalization, and was correlated with an increase in migration and invasion [567 mutants exhibit higher collagenolytic activity and three-dimensional growth within a collagen matrix, thereby promoting enhanced matrix invasion in ovarian cancer cells [567 impacted the integrity of cell monolayer, cell motility and multicellular aggregate dynamics in ovarian cancer cells, promoting metastasis-associated behaviors [573 influenced cell migration, suggested by the ability of a phospho-defective mutant to inhibit migration of cells endogenously expressing MMP14 [573 prompted internalization of MMP14 together with pericellular collagen, establishing an environment for expansive growth in three–dimensional collagen matrix, whereas lack of responding to EGFR signaling triggered invasive growth. Therefore, the phosphorylation of Tyr573 modulates cell surface dynamics of MMP14, thereby regulating the transition between invasive and expansive growth [MMP-14 phosphorylation is known to play an important role in fine-tuning the activity of MMP14 at the cell surface and in the activation of intracellular signaling, and it is critical for regulating the pro-metastatic function of this metalloproteinase. This membrane-type metalloprotease has nine potential phosphorylation sites in its multidomain structure  are tyrosine phosphorylated by the extracellular vertebrate lonesome kinase (VLK). These phosphotyrosines are found exclusively within the hemopexin domains of the MMPs. Interestingly, proteins distantly related to MMPs, which contain hemopexin-like domains, were found to be tyrosine phosphorylated at identical positions by VLK. The structural conservation of these phosphorylation sites suggests that they play a role in regulating MMP activity, a potential function, which still remains largely undiscovered ,93. Addiectively ,95,96.N-acetylglucosamine (GlcNAc) dimers, which in most cases are attached to a cell-membrane associated core protein. During GAG-chain synthesis HS undergoes several sulfotransferase-mediated N- and O-sulfations. These modifications do not undergo completion, which results in strongly negatively charged sugar chains with variable length and degree of sulfation interrupted by stretches of unmodified, neutral regions. The sulfated regions bind basic peptide motifs of many signaling molecules, including morphogens , cytokines, and most chemokines [An additional layer of regulation of extracellular proteolysis is mediated by interactions of MMPs with glycans in the extracellular space rather than direct glycosylation or phosphorylation of the protease or substrate. Glycosaminoglycans (GAGs) are extracellular glycans, which are composed of linear, unbranched repeats of disaccharide units. Heparan sulfate (HS) is a GAG-family member expressed by virtually every cell of a multicellular organism composed of repeats of glucoronic acid (GlcA) and emokines ,98,99. Temokines ). Examplemokines ,103,104.emokines ,107,108.N-glycans, GAGs can modulate the stability of substrates. FGF2 binding to heparin, an extremely sulfated HS variant expressed by connective-tissue type macrophages, stabilizes FGF2 and protects it from degradation by proteolysis [Drosophila in vivo [Signaling processes in the extracellular space usually involve at least one form of substrate proteolysis. Therefore, co-factors have to act as decision makers to ensure regulated processing of signaling proteins. Multiple cases have been reported, where this is mediated by substrate binding to GAGs. For example, similar to teolysis ,110. Theteolysis ,112. Desteolysis . Here, Hteolysis . Further in vivo ,115. An  in vivo .GAGs also regulate extracellular proteolysis at the protease level by modulating activation, localization, and protease–substrate interactions  middle. MMP biology has been revolutionized with the recognition of extracellular proteolysis not as a simple mechanism of ECM degradation but as a regulatory mechanism for precise cellular control of biological processes. The paradigm shift for MMP functions from ECM degradative proteases to important regulators of essential cellular processes has highlighted the physiological relevance of these proteases, directly implicated by the relationship between MMP expression and disease development ,8. Hence