Currently, no specific biochemical markers can either predict progression to psoriatic arthritis or response to therapies. This study aimed to identify osteoimmunological markers applicable to clinical practice, giving a quantitative tool for evaluating pathological status and, eventually, to provide prognostic support in diagnosis. (2) Methods: Soluble (serum) bone and cartilage markers were quantified in 50 patients with only psoriasis, 50 psoriatic patients with psoriatic arthritis, and 20 healthy controls by means of multiplex and enzyme-linked immunoassays. (3) Results: Differences in the concentrations of matrix metalloproteases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), receptor activator of nuclear factor kappa-B- ligand (RANK-L), procollagen type I N propeptide (PINP), C-terminal telopeptide of type I collagen (CTx-I), dickkopf-related protein 1 (DKK1), and sclerostin (SOST) distinguished healthy controls from psoriasis and psoriatic arthritis patients. We found that MMP2, MMP12, MMP13, TIMP2, and TIMP4 distinguished psoriasis from psoriatic arthritis patients undergoing a systemic treatment, with a good diagnostic accuracy (Area under the ROC Curve (AUC) > 0.7). Then, chitinase-3-like protein 1 (CHI3L1) and MMP10 distinguished psoriasis from psoriatic arthritis not undergoing systemic therapy and, in the presence of onychopathy, MMP8 levels were higher in psoriasis than in psoriatic arthritis. However, in these latter cases, the diagnostic accuracy of the identified biomarkers was low (0.5 < AUC < 0.7). (4) Conclusions. By highlighting never exploited differences, the wide osteoimmunological biomarkers panel provides a novel clue to the development of diagnostic paths in psoriasis and psoriasis-associated arthropathic disease. Adult psoriatic disease depicts a continuum encompassing disease progression from psoriasis (Ps) to psoriatic arthritis (PsA) . In contA biochemical marker that clearly predicts PsA in a cohort of Ps patients is still elusive. A biomarker is defined as a measurable characteristic indicating a biological or pathophysiological process and can be used to identify the risk to develop a certain disease . SeveralBased on this background, this study aimed to investigate the possible association between a wide panel of osteoimmunological biomarkers with Ps and PsA, and in the relative sub-cohorts in order to identify a possible laboratory tool that could support PsA diagnosis and early prediction.2, respectively.In both Ps and PsA groups, a prevalence of male subjects, 66% and 78%, respectively, was observed despite no gender prevalence being reported. Median ages were 48 (19–82) years in Ps, 51(28–79) years in PsA, and 48 (29–67) years in controls. The body mass index (BMI), in the three groups, was 25 (23–29), 26 (23–28), and 24 (22–25) kg/mThe Psoriasis Area Severity Score (PASI) was higher in Ps (6 (5–12)) than in PsA (4 (1–7)). Onychopathic signs were present in 38% of Ps subjects and 48% of PsA subjects, according to the literature ,12, PsA When considered in their entirety, without any subgrouping for treatment status, no significant differences were observed for any of the tested markers between Ps and PsA. Conversely, compared to the control group (CTRL), both Ps and PsA groups differed for most of the analyzed molecules, except for MMP3, MMP7, MMP12, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, OPG, C-telopeptide of type II collagen (CTx-II), and chitinase-3-like protein 1 (CHI3L1) .Furthermore, statistically significant correlations were found between markers’ concentration and duration of both Ps and PsA: MMP2, MMP12, MMP13, TIMP1, TIMP2, TIMP3, sclerostin (SOST), and CHI3L1 in Ps (positive correlation) and with MMP10 and TIMP2 in PsA (negative correlation). Moreover, in Ps group, MMP8, MMP10, and CTx-I positively correlated with PASI score, while TIMP4 was negatively correlated .The Ps and PsA cohorts were further divided based on the therapy regimen (subjects undergone to systemic treatments (ST) and not systemically treated (NST)).n = 19 and n = 17, respectively) were compared, MMP2 , MMP12 , MMP13 , TIMP2 , and TIMP4  were higher in Ps to PsA (p = 0.004).When Ps and PsA subjects ST (s to PsA A. As expThe Relative Operating Characteristic (ROC) analysis shows that the area under the ROC curve (AUC) for both the single markers  and their combination (0.755 to 0.845) display a moderately accurate diagnostic potential in discriminating Ps from PsA ST patients. Noteworthy, the combination of all these markers gave the highest diagnostic accuracy (AUC = 0.845) .n = 31) and PsA NST (n = 33) significantly differed for MMP10 , and CHI3L1  (p = 0.002).By focusing on the NST group, Ps NST (= 0.042) B, with tp = 0.011), and negatively with PsA duration .In Ps NST, MMP10 positively correlated with CHI3L1 , Ps ST (n = 19) had significantly increased serum levels of MMP2 , MMP3 , MMP12 , MMP13 , TIMP2 , TIMP3 , and CTx-I   .n = 17) vs. PsA NST (n = 33).Interestingly, no osteoimmunological biomarkers were statistically different in the comparison PsA ST (p = 0.009) and higher circulating MMP8  compared to onychopathic PsA patients (PsA O) (Ps patients with onychopathy (Ps O) had higher PASI (6.5 vs. 3.7  (PsA O) . ROC ana (PsA O) .p = 0.002). Non-onychopathic Ps compared to PsA counterparts achieved significance only for PASI  gave a good diagnostic accuracy. Consequently, this panel is particularly promising because of the wide use of MTX in both Ps and PsA. The effects of systemic treatments on the osteoimmune function is clearly depicted, in our study, by the decrease in the circulating levels of most of the markers here considered. A second set of biomarkers seems to be useful in differentiating between Ps and PsA that have never undergone systemic treatments (NST). Given their naïve condition, the comparison of Ps NST and PsA NST patients could be considered as the most useful. This set comprises of MMP10 and CHI3L1 whose levels are, moreover, reciprocally correlated. CHI3L1 is an inactive chitinase due to the lack of the catalytic domain whose physiological functions have not been fully clarified, although it may play a role in inflammation ,18,19. BNail psoriasis is, among others, a risk factor for the development of PsA, especially within the distal interphalangeal joints, as suggested by McGonagle and colleagues ,25. RemaIn those subjects presenting onychopathy signs, MMP8 concentrations were higher in Ps than in PsA. Being a collagenase, MMP8 preferentially cleaves the interstitial type I collagen and is the first collagenase that appears during dermal wound-healing ,32. MMP8The cross-sectional nature of this study and the somehow limited sample size (pilot study) represent the main limitations, although this is compensated by the great homogeneity of the selected cohorts that limits the number of variables potentially affecting the circulating levels of the analyzed biomarkers. Remarkably, due to the exiguity of the sample, it was not possible to evaluate the influences of drugs and severity on the biomarker’s levels. Future studies are mandatory to assess the osteoimmunological biomarker specificity for PsA and further compare the present findings with other autoimmune diseases involving joints, such as RA or gout.clinicaltrials.gov (NCT03455166). The study was carried out following the rules of the Declaration of Helsinki of 1975, revised in 2008 .The clinical trial was approved by the ethical committee , and registered at A written consent to the use of clinical data was obtained from all patients after being informed about the study procedures, their benefits, and eventual risks.n = 50) and patients with Ps and PsA  were enrolled at the department of dermatology and venereology of the IRCCS Istituto Ortopedico Galeazzi , starting from June 2015 until April 2017, during routine clinical activities. Inclusion criteria for Ps patients were plaque psoriasis  for more than six months, neither current nor previous treatment with biological drugs, and no joint involvement.Patients with a diagnosis of plaque psoriasis  criteria  were actPsoriatic onychopathy was included only if nail psoriasis severity index (NAPSI)  ≥ 3 poinExclusion criteria, common to the two groups, included pregnancy, current or previous malignancies, other acute or chronic inflammatory diseases, infectious diseases (human immunodeficiency virus (HIV), hepatitis B and C , tubercuDuring the outpatient visit, Ps history, disease onset and evolution were evaluated. The most prevalent type of Ps was determined, along with the body sites involved and the presence of the onychopathic trait. A psoriasis area and severity index (PASI) score was assigned by two independent board-certified dermatologists. PsA diagnosis was formulated by two independent board certified rheumatologists prior to the dermatological examination, based on anamnestic and clinical evaluations of the patient’s inflammatory and imaging indexes. In particular, based on CASPAR criteria  and ultrFinally, laboratory analyses completed the diagnostic path by investigating markers useful in the differential diagnosis with other forms of inflammatory arthritis .® . Blood was immediately centrifuged at 1300× g  and serum was aliquoted and stored at −80 °C until assayed. Serum samples from 20 healthy Caucasian age- and sex-matched subjects  were used as a control (CTRL).Blood samples were collected by standard venipuncture of the antecubital vein in SST II Advance Vacutainer® Multiplex System .Serum matrix metalloproteinases  and their tissue inhibitors  were quantified using a multiplex assay on a Bio-Plex©, Houston, TX, USA). Serum markers of cartilage degradation and inflammation, CTx-II and CHI3L1, also known as YKL-40, were measured by a competitive enzyme immunoassay (Cloud-Clone Corp©) and a sandwich enzyme immunoassay (BioVendor-Laboratorni Medicina A.S.), respectively. Inhibitors of the Wnt signaling pathway, DKK1, and SOST, were assayed in serum by a solid-phase ELISA .Osteoimmune markers, OPG, and RANKL, concentrations were measured in serum using monoclonal antibody-based immunoassays (ELISA) . A marker of bone resorption, CTx-I, and a marker of bone formation, PINP, were measured in serum by monoclonal antibody-based immunoassays (Cloud-Clone Corpw) and inter-assay (CVb) variations were: 2.5–4.9% and 1.7–9.0% for OPG, 7.25–11.51% and 11.21–12.77% for RANKL, <10% and <12% for CTx-I, PINP, CTx-II, CHI3L1, 4.2% and 7.6% for DKK1, 2.1% and 10.8% for SOST.The lower limits of detection (LOD) were 0.03 pmol/L for OPG, 0.4 pmol/L for RANKL, 44.3 pg/mL for CTx-I, 12.4 pg/mL for PINP, 52.3 pg/mL for CTx-II, 5.0 pg/mL for CHI3L1, 4.20 pg/mL for DKK1, and 1.78 pg/mL for SOST. Intra-assay  in order to limit variability in the final analytical output ,13.Shapiro–Wilk’s normality test was performed on data from the entire cohort. Since the non-parametrical distribution of the values, quantitative parameters are expressed as the median and the interquartile range in the descriptive analysis. The within-group comparisons were performed using Mann–Whitney U-test. Kruskall–Wallis test was used for multiple comparison. Spearman’s rank correlation test was applied to evaluate correlations that were considered significant when r ≥ 0.25. Diagnostic accuracy of those markers displaying a statistically significant difference in the subgroup’s comparison was determined by the ROC (receiver operating characteristic) curves analysis. The AUC (area under curve)-based accuracy was classified according to Swets JA .p < 0.05. Analyses were performed using SPSS software .The significance was set at This study, comparing a large panel of osteoimmunological biomarkers, highlighted profound differences between Ps, PsA, and healthy controls. These phenotypic differences, which are attenuated in the comparison between Ps and PsA, reinforces the thesis according to which these two diseases belong to the same pathological spectrum. However, we also identified important differences in the expression of specific tissue-remodeling associated enzymatic activities in selected sub-cohorts of Ps and PsA subjects that seem to be dependent upon the treatment (systemic vs. topic) and the presence nail involvement (onychopathy).


Mitogen-activated protein kinases (MAPKs) are intracellular molecules regulating a wide range of cellular functions, including proliferation, differentiation, apoptosis, cytoskeleton remodeling and cytokine production. MAPK activity has been shown in normal kidney, and its over-activation has been demonstrated in several renal diseases. The extracellular signal-regulated protein kinases  signalling pathway is the first described MAPK signaling. Intensive investigations have demonstrated that it participates in the regulation of ureteric bud branching, a fundamental process in establishing final nephron number; in addition, it is also involved in the differentiation of the nephrogenic mesenchyme, indicating a key role in mammalian kidney embryonic development. In the present manuscript, we show that ERK1,2 signalling mediates several cellular functions also in mature kidney, describing its role along the nephron and demonstrating whether it contributes to the regulation of ion channels and transporters implicated in acid-base and electrolytes homeostasis. The MAPK cascade is a signal transduction common to several cell types, and it is involved in many biological processes, including cell proliferation, differentiation, development, and apoptosis . The ERK1,2 signalling is the first identified MAPK signalling . The typMAPK/ERK1,2 signalling has been shown to exert a significant role in renal morphogenesis and differentiation [The activation of MAPK/ERK1,2 has been recently reported in both nephron progenitors (NPs) and developing kidney. Chemical MEK inhibition in rat embryos has been shown to affect nephrogenesis [ntiation . The actogenesis , and NPsogenesis .MAPK signalling is activated by a wide range of stimuli also in mature kidney; these pathways have been shown to be critical in mediating cell response to different types of stress and injuries and to play a role in the pathophysiology of several diseases. Oxidant-induction of ERK1,2 signalling was demonstrated under ischemic injury, with ERK1,2 inhibition showing a favorable effect on the ischemia/reperfusion renal damage . In addiThe PT has a key role in acid base, water, electrolytes and nutrients balance, by reabsorbing 60–70% of filtered NaCl, the bulk of filtered bicarbonate and almost all nutrients . It is a3−); (2) by secreting protons (H+); (3) by synthetizing the major urinary buffer (NH3/NH4+) and new molecules of HCO3− through glutamine metabolism and (4) by reabsorbing filtered citrate, which equals alkali reclamation [+ secretion are fulfilled by a synergistic mechanism. H+ ions are mainly secreted into the lumen through the electroneutral Na+/H+ antiporter NHE3, expressed on the apical membrane. Secreted H+ combines with filtered HCO3− into the luminal fluid, a reaction that is catalyzed by the luminal carbonic anhydrase (Ca IV), generating carbonic dioxide (CO2), which diffuses through the membrane into the cells, reconstituting H+ and HCO3− [3− molecules [+-citrate cotransporter, NaDC1, is part of the adaptive response to an acid load [The PT contributes to the maintenance of acid-base homeostasis through multiple tasks: (1) by reclaiming almost all filtered bicarbonate  phosphorylation, with a similar time course, suggesting that media acidification activates the typical Raf1/MEK/ERK1,2/p90RSK axis. Either Raf1, MEK and p90RSK inhibition blocked acid-induced NaDC1 activity in OKP cells. Taken together, these studies suggest that ERK1,2 signalling is activated by media acidification and an acid loading in vitro and in vivo respectively, and that the signalling mediates several adaptive responses to acidosis, namely increased NHE3 and NaDC1 activities , a cellular model of PT . In thei rodents , further rodents . Specifitivities . 2+) reabsorption and urine concentration [The loop of Henle is a peculiar nephron site with a characteristic anatomic configuration and specific properties. In normal subjects, the TAL reabsorbs 25–30% of filtered NaCl and plays a crucial role in calcium , in parallel, K+ recycles into the lumen via the K+ channel (ROMK) [+ ions are pumped through the Na+/K+-ATPase system and a conductive Cl− exit is mediated by a Cl– channel. NKCC2 and in particular ROMK generate the “driving force” for paracellular reabsorption of Na+, K+, Ca2+, and Mg2+ [2+ absorption, inhibiting ROMK activity and influencing the tight junction (TJ) structure [These functions are realized through the coordinated activity of several transporters and channels. Filtered NaCl enters the cells thanks to the presence of the luminal Nal (ROMK) . On the and Mg2+ ,23,24. Ttructure ,26,27.To date, ERK1,2 signalling pathway activation has been described in intact renal tubular cells of the TAL ,29,30.In 1995, Terada et al.  showed tIn response to hypertonic stress, renal medullary epithelial cells modify their gene expression through the activation of the MAPK cascade ,33, and Gallazzini et al. examined the mechanisms by which tonicity regulates ROMK gene transcription in rat MTAL and in cultured mouse TAL cells . In MTAL3− absorption thanks to the activity of the apical membrane Na+/H+ exchanger [3− absorption in the MTAL is mediated by different signalling pathways [The MTALs participate also in the regulation of acid-base balance through luminal HCOxchanger ,37. The pathways  with no pathways ,40.Finally, the distal nephron and particularly the medullary TAL segment plays an important role in response to the ischemic or acute renal damage. It has been demonstrated that after toxic ischemic renal injury, TAL cells show an altered expression/activation of many genes ,42. In tRecent studies analysed in vitro the effect of several growth factors on human renal thick ascending limb and distal convoluted cells (TALDC) proliferation, demonstrating a significant, dose- and time-dependent phosphorylation of ERK1/2 after stimulation with EGF . In cont2+ and Mg2+ handling [The DCT plays a key role in NaCl reabsorption and Cahandling ,49.+ and plays a key role in blood-pressure regulation [Here, the sodium-chloride cotransporter (NCC), localized at the apical membrane, is responsible for the reabsorption of 5–10% of filtered Nagulation . Indepengulation . In theigulation , while Pgulation . This wa+2 and phosphate (PO4−2) homeostasis through direct actions on the PT and the DCT [+2 entry in mDCT only [Parathormone (PTH) and Vitamin D are known to control Ca the DCT ,54. Ko a the DCT . AdditioDCT only .Both cell surface and total NCC expression are inhibited by the serine/threonine with no lysine kinase WNK4, via ERK1,2 signalling. Hypertonicity as well as EGF stimulated ERK1,2 phosphorylation in mDCT cells; these responses were totally blocked in the presence of mutations of WNK4, resulting in pseudo-aldosteronism type 2(PHA II), a rare form of hypertension associated with hyperkalemia ,57. NCC 2+ entry is mediated by the transient receptor potential vanilloid 5 (TRPV5), expressed across the apical membrane, while it is extruded by the basolateral Na+/Ca2+ exchanger (NCX1) [2+ transport in DCT cells but is dispensable for the release of Ca2+ in the PT [In the DCT, Car (NCX1) . Sneddonn the PT .2+ and plays a pivotal role in renal Mg2+ handling [2+ uptake in mDCT cells, by inhibiting the ERK1,2 signalling pathway [The DCT absorbs 10–15% of the filtered Mghandling . Cyclosphandling . Kim et  pathway .2+, but also Ca2+ [2+ and decreased serum Mg2+ levels. In this inherited disease, the altered EGF leads to decreased activity and membrane expression of TRPM6, resulting in a decrease of Mg2+ reabsorption in the distal convoluted tubule [TRPM6 is a cation channel which transports mainly Mglso Ca2+ , exclusilso Ca2+ . Indeed,d tubule .+ absorption through the amiloride sensitive-Epithelial Sodium Channel (ENaC) expressed at the apical membrane and regulated by aldosterone plasma levels [+ absorption via ENaC is coupled to K+ secretion through the renal outer medullary potassium channel (ROMK) due to lumen electro-negativity [+ absorption [+-ATPase and the basolateral Cl−/HCO−3 exchanger (AE1). In β-ICs, H+-ATPase is expressed on the basolateral membrane, while the Cl−/HCO3− exchanger (Pendrin) on the apical membrane.The CD consists of two types of cells: Principal cells (PCs) and α-β- intercalated cells (ICs). PCs mediate Naa levels . Na+ absgativity ,74. ICs sorption . α-ICs a+) administration induces morphological alteration of CD and downregulation of AQP2 in rat kidney medulla. In this context, rat inner medullary collecting duct (IMCD) phospho-proteomic profile was examined after acute lithium (Li) administration [+ exposure, increased phosphorylation in activation sites of ERK1,2 and p38 were observed. Since pretreatment with the MAPK inhibitor abolished Li+-induced upregulation of pSer261-AQP2, ERK1,2 and p38, they have been suggested as early targets of Li+, possibly playing a role in the onset of Li+-induced polyuria [+-treatment, ERK1,2 dysregulation was associated with a dysregulation of GSK3-beta [Interestingly, chronic lithium . In-vitro studies were conducted using primary and immortalized murine renal-collecting duct cells (mCT12); the cells were exposed to EGF, ATP, phorbol 12-myristate 13-acetate (PMA) or 2,5-di-tert-butyl-hydroquinone (DBHQ). In all conditions, increased ERK1,2 phosphorylation was observed, and ERK1,2 inhibition with PD98059 reduced EGF, ATP, and PMA-dependent inhibition of amiloride-sensitive Na+ absorption. ERK1,2-mediated inhibition of Na+ absorption was associated with phosphorylation of β- and γ-ENaC subunits, on the C-terminus [sc) without affecting the surface expression of ENaC [The EGF exhibits a negative effect on amiloride-sensitive Nasorption . Cotton’terminus . Residue of ENaC ,82, comp of ENaC . Interes of ENaC .+ channels are expressed at the apical membrane of the CD, including ROMK, Ca2+-activated big-conductance K+ channels (BK) and K+ Two Pore Domain Channel Subfamily K Member 1 (KCNK1). ROMK and BK channels have been claimed as the main K+ channels [+ intake induced decreased ERK1,2 and p38 phosphorylation, resulting in increased BK channel activity [+ channels in CCD, ROMK-like small-conductance K+ channels (SK) and BK channels. PGE2, also known as dinoprostone, is a potent inflammatory mediator produced by cyclooxygenase 2 (COXII). In this study, PGE2 dramatically increased Protein kinase C (PKC)-dependent phosphorylation of ERK1,2 and p38. The effect of PGE2 was EP1 receptor-specific. Increased COXII expression and PGE2 production were induced by low-potassium intake. Even though the effect of K+-depletion on PGE2 production depends on the duration of diet and animal species, increased PGE2 level during K+-restriction might have a regulatory role on apical K+ channels in CCD through MAPK pathway and PKC [+ secretion through the phosphorylation of p38 and ERK, and the stimulation of protein tyrosine kinase (PTK) in CCD [Different types of Kchannels . Li and activity . Jin et activity  reported and PKC . In addi) in CCD ,88.+-K+-ATPase in rat CCD β-ICs, demonstrating the role of β-adrenergic receptors and Ras-Raf-1-ERK1,2 signalling [Laroche-Joubert and colleagues studied the signalling pathway-mediating isoproterenol-dependent stimulation of Hgnalling . Recentlgnalling . In thisMagnesium is crucially involved in several vital biological processes such as the regulation of enzymes and ion channels activity and the stabilization of ATP, ADP, RNA, and DNA. Severe magnesium deficiency can cause tetany, cardiac arrhythmia, and bone instability. As previously described, ERK 1,2 inhibition may lead to hypomagnesemia ,69,70,712+ level should be regularly monitored to prevent potentially fatal complications. More studies are expected to address this relevant focus.In conclusion, we propose that in patients treated with calcineurin inhibitors  and with drugs interfering with ERK1,2 signalling, serum MgIt has been widely reported that ERK 1,2 activation is an important pathway sustaining podocyte injury in several proteinuric nephropathies. In fact, MAPK/ERK signalling pathway has been found to be activated in the mesangium of proteinuric patients . In part+ retention-related hypertensive mechanisms [+ transporters and channels, as NCC and ENaC. Whether hypertension, a common side effect of drugs inhibiting BRAF, may be the consequence of the loss of this inhibition, requires additional studies.Blood pressure maintenance is mostly dependent on sodium balance, thus alterations of renal sodium handling are a contributing mechanism into the pathogenesis of salt-sensitive hypertension. RAAS is known to be the most important promoter of this condition. Recent reports demonstrated that all prorenin, renin and angiotensin II, and aldosterone are able to induce ERK 1,2 phosphorylation, and this might potentiate tubular Nachanisms . Moreovechanisms . Howeverchanisms . Of notechanisms . HoweverThe activation of the ERK1,2 signalling pathway is the result of several proliferative signals, however, it is not surprising that its overactivation has been found in many forms of cancer. MAPK pathway is estimated to be deregulated in almost 50% of human malignancies, activating mutation of BRAF in 5–10% of cancers, especially hairy cell leukemia, melanoma, colorectal cancer, papillary thyroid cancer, non-small cell lung cancer (NSCLC), and ovarian cancer . Thus, tAs described in the previous paragraphs, the ERK 1,2 signalling pathway is involved in acid-base regulation due to its influence on several tubular transporters. Specifically, it has been widely demonstrated in numerous in vitro and in vivo studies that the ERK 1,2 pathway is activated by intracellular acidosis and mediates compensatory responses ,15,16,17ERK1,2 signalling pathway is known to be critical in embryonic kidney development. The present review shows that it mediates the transcriptional and post-transcriptional regulation of several nephron channels and transporters in mature organs. The pivotal role in kidney homeostasis is further demonstrated by the over-activation in several renal diseases. The clinical use of molecules targeting ERK1,2 signalling, especially in oncology, is expected to influence numerous vital nephron functions. In conclusion, a close monitoring of acid-base and electrolytes balance parameters, together with glomerular function markers, should be a routine part of clinical care in patients treated with drugs interfering with ERK1,2 signalling pathway.


Jatropha, we found six members of the TPP family. Five of them JcTPPA, JcTPPC, JcTPPD, JcTPPG, and JcTPPJ are highly expressed in female flowers or male flowers, or both, suggesting that members of the JcTPP family may participate in flower development in Jatropha. The wide expression of JcTPPJ gene in various organs implied its versatile roles and thus was chosen for unraveling its biological functions during developmental process. We constructed an overexpression vector of JcTPPJ cDNA driven by the cauliflower mosaic virus (CaMV) 35S promoter for genetic transformation. Compared with control Arabidopsis plants, 35S:JcTPPJ transgenic Arabidopsis plants presented greater sucrose contents in their inflorescences and displayed late-flowering and heterostylous phenotypes. Exogenous application of sucrose to the inflorescence buds of wild-type Arabidopsis repressed the development of the perianth and filaments, with a phenocopy of the 35S:JcTPPJ transgenic Arabidopsis. These results suggested that the significantly increased sucrose level in the inflorescence caused (or induced) by JcTTPJ overexpression, was responsible for the formation of heterostylous flower phenotype. However, 35S:JcTPPJ transgenic Jatropha displayed no obvious phenotypic changes, implying that JcTPPJ alone may not be sufficient for regulating flower development in Jatropha. Our results are helpful for understanding the function of TPPs, which may regulate flower organ development by manipulating the sucrose status in plants.Trehalose-6-phosphate (T6P) phosphatase (TPP), a dephosphorylating enzyme, catalyzes the dephosphorylation of T6P, generating trehalose. In  Trehatebrates ,3,4,5. Ttebrates . Trehalatebrates ,6. ArabiTRE gene ,8. The Tg plants ,10,11,12AtTPS1 gene from Arabidopsis, which encodes a T6P synthase. As an endogenous substance, trehalose, along with TRE, regulates carbohydrate allocation in Arabidopsis [Arabidopsis, transgenic plants overexpressing bacteria trehalose 6-phosphate synthase (otsA) display small, dark green leaves and early-flowering phenotypes [Arabidopsis plants overexpressing bacteria trehalose 6-phosphate phosphatase (otsB) display relatively larger, lighter green leaves and late-flowering phenotypes [RAMOSA3 (RA3) encodes a TPP enzyme that controls maize inflorescence architecture by mediating the axillary meristems via modulation of trehalose and T6P levels [ra3 mutants display long branches or increased numbers of floral meristems [The presence of trehalose in a wide variety of organisms suggests a pivotal and ancient role concerning trehalose metabolism in the plant kingdom. Blázquez et al.  isolatedbidopsis ,15, cellbidopsis , embryo bidopsis ,18, leafbidopsis ,20, the bidopsis , the phobidopsis , and abibidopsis ,26,27,28enotypes ; howeverenotypes . In maizP levels . Owing teristems .OsTPPs improves grain yield under well-watered growth conditions and mild drought conditions, and significantly increases grain yield under severe drought conditions at the highly susceptible flowering stage [OsTPP7 enhances anaerobic germination by promoting starch mobilization to drive the growth kinetics of both germinating embryos and elongating coleoptiles [AtTPPD results in more tolerance to high-salinity stress, whereas loss-of-function in AtTPPD causes hypersensitivity to salinity stress [AtTPPG also involves in abscisic acid (ABA) related stomatal closure [Because of its non-reducing feature and the stability of the α,α-1,1 linkage, trehalose might be involved in the response to stress . In riceng stage . Overexpeoptiles . Overexpy stress . AtTPPG  closure .Arabidopsis lines overexpressing maize Sucrose non-fermenting-1 (SNF1)-related protein kinase 1 (ZmSnRK1.1), ZmSnRK1.2, or ZmSnRK1.3 accumulate high sucrose content in rosette leaves and mature seeds, and display a shortened flowering time in ZmSnRK1.1 lines and a prolonged flowering time in ZmSnRK1.2 and ZmSnRK1.3 lines, respectively. In Arabidopsis, SnRK1 positively regulates FUSCA3 (FUS3) stability and INDETERMINATE DOMAIN 8 (IDD8) phosphorylation to mediate flowering time; FUS3 encodes a B3-domain transcription factor regulating organ  formation and developmental phase transition, and IDD8 regulates sucrose synthesis (SUC) gene expression to affect photoperiodic flowering [Cucumis sativus), downregulation of sucrose transporter (CsSUT1) can decrease sucrose content in male flowers, and perturb male flower development generating smaller flower [Sugar sensing and signaling may be involved in various aspects of growth and development in higher plants ,32. Wanglowering ,35,36,37r flower .Jatropha curcas L., belonging to the Euphorbiaceae family, is a perennial woody plant and widely distributes in tropical and subtropical regions. Jatropha has a dichasial cyme inflorescence bearing 0–10 female and 25–215 male flowers [Jatropha is considered as a promising renewable energy plant [JcTPPJ gene from Jatropha and overexpressed it in Arabidopsis and Jatropha for its functional validation. The transgenic Arabidopsis plants displayed heterostylous flowers and late-flowering phenotypes; however, the transgenic Jatropha plants did not display obvious phenotype change. flowers . Owing tgy plant ,44,45,46JcTPPA, JcTPPC, JcTPPD, JcTPP1, JcTPPG, and JcTPPJ that are homologous to Arabidopsis trehalose-6-phosphate phosphatase (AtTPP) genes were identified from Jatropha. JcTPPs contain a HAD-like  domain similar to that in Escherichia coli trehalose 6-phosphate phosphatase (otsB) [A total of six genes e (otsB) ,48. PhylJcTPPs was further investigated in different tissues, including roots, stems, young leaves, mature leaves, stem apices, female flowers, male flowers, and fruits using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The JcTPPJ gene was broadly expressed in all these tissues, and particularly high expressed in roots, stems, and male flower tissues  35S promoter were obtained. Transgenic Arabidopsis plants were selected in the presence of kanamycin, and the expression level of JcTPPJ was validated via qRT-PCR  Arabidopsis with 50 mM sucrose and trehalose solutions, respectively. Both treatments suppressed the development of the perianth and stamen filaments, and the sucrose treatment caused a more obvious phenotype  for preventing self-pollination and promoting outcrossing, which aids in environmental adaption and species diversity ,54,55,56rphology , which sTPP can promote growth and varying severity of morphological abnormalities in several species [Arabidopsis plants overexpressing AtTPPB, AtTPPC, and AtTPPI is relatively short, preventing the protection and covering of the pistil and stamen by the perianth [JcTPPJ in Arabidopsis results in phenotypes similar to those of AtTPPB, AtTPPC, and AtTPPI transgenic plants; however, the filaments of the stamen are relatively short in 35S:JcTPPJ transgenic Arabidopsis, which implies that JcTPPJ plays a stronger role in preventing the development of male floral organs than does AtTPPB, AtTPPC, and AtTPPI as assessed by their overexpression in transgenic plants  catalyzes dephosphorylation of T6P to generate trehalose [TPP1 driven by the OsMads6 promoter in maize increases the sucrose content in ear spikelets. Kretzschmar et al. [OsTPP7 activity can enhance sucrose availability. In Arabiodpsis, the sucrose content is slightly lower in tppj mutants than in WT plants [E. coil otsB and AtTPPs in Arabidopsis results in a late-flowering phenotype, respectively [Arabidopsis plants overexpressing SNF1 KINASE HOMOLOG 10 (AKIN10) encoding a sucrose non-fermenting-1 (SNF1) related protein kinase, displayed phase transition, and organ development defects by delaying the degradation of FUS3 [JcTPPJ in Arabidopsis also resulted in increased sucrose contents, which may cause the development of short perianth and stamen filaments and a delay in flowering  in which lighting is provided by cool-white fluorescent bulbs. Mature Jatropha plants were transplanted into an experimental field at the Xishuangbanna Tropical Botanical Garden , Chinese Academy of Sciences [−2 s−1 in the summer and 1550 μmol m−2 s−1 in the winter [Arabidopsis seeds were germinated on half-strength Murashige and Skoog medium (1/2 MS). First, the seeds were vernalized for 2 days at 4 °C, and then were transferred to program ray radiation incubator  under a long-day (16 h light/8 h dark) photoperiod with 22 °C/20 °C day/night temperatures for seven days. The seedlings were transplanted to peat-based soil and maintained in program ray radiation incubator. We collected the roots, stems, mature leaves, young leaves, stem apices, male flowers, female flowers, and fruits of WT Jatropha; the young leaves of transgenic Jatropha; and the rosette leaves of transgenic Arabidopsis for qRT-PCR, all of which were immediately frozen in liquid nitrogen and then stored at −80 °C until use.The cultivar ‘Flowery’ of Sciences . The phoe winter . The AraArabidopsis plants and heterozygous (T0 and T1) Jatropha plants were analyzed. When the main inflorescence shoot had elongated past 1.0 cm, we recorded the flowering time and counted the numbers of Arabidopsis rosette leaves. Twenty plants were used to investigate genotypes. For the flower phenotypes, the flower buds were dissected for microscopic analysis .The phenotypes of homozygous (T3) JcTPPJ gene, total RNA was extracted from mature leaves of Jatropha with a pBIOZOL RNA extraction reagent , and the quality of RNA was measured by a NanoDrop 2000 spectrophotometer . In accordance with the TAKARA PrimeScript™ RT Reagent Kit , we used approximately 1.0 µg of total RNA for cDNA synthesis. The full-length coding sequence of JcTPPJ was obtained from this cDNA library by PCR with TransStart KD Plus DNA Polymerase  and gene-specific primers. A combination of XC9 and XC10 primers were used  to perform sequence alignment, and manually edited using BioEdit software (https://bioedit.software.informer.com/). MEGA (version 5.0) software (http://www.megasoftware.net/) was used to construct a phylogenetic tree based on the N-J method with 1000 bootstrap replications; the bootstrap percentages were shown on the dendrogram branch points. The sequences of JcTPS1 and AtTPS1 were used as outgroups.The full-length protein sequences of JcTPPA, JcTPPC, JcTPPD, JcTPP1, JcTPPG, JcTPS1, AtTPS1, and AtTPPA-J and bacteria otsB were downloaded from the NCBI GenBank . We then35S:JcTPPJ plant overexpression vector. The confirmed cDNA sequence was excised from the sequenced vector using appropriate restriction enzymes and then cloned into a pOCA30 binary vector with the CaMV 35S promoter. The vector was introduced into Agrobacterium tumefaciens strain EHA105 via the freeze–thaw method [Arabidopsis plants were transformed by the floral dip method [Jatropha was performed using Agrobacterium-mediated methods described as Fu, et al. [Jatropha plants were confirmed by genomic PCR and qRT-PCR methods.We constructed a w method , and Colp method . To iden, et al. . TransgeJcTPPJ and identify positive 35S:JcTPPJ transgenic plants, qRT-PCR was performed using SYBR® Premix Ex Taq™ II  on a LightCycler 480II  device. The JcGAPDH [AtActin2 genes were used to normalize the transcript levels of specific genes of Jatropha and Arabidopsis, respectively. At least three biological replicates were used for all samples. The 2Ct−ΔΔ method was used to analyze the data described as Livak and Schmittgen [Ct value was from difference between Ct values of reference gene JcGAPDH or AtActin2 and each target gene; the ΔΔCt was calculated by subtracting the mean ΔCt of the control samples from mean ΔCt of transgenic samples. Primers for qRT-PCR are listed in To investigate the expression patterns of  JcGAPDH  and AtAchmittgen . The ΔCtv/v) Tween-20 . For sucrose or trehalose treatment experiment, 10 μL of the working solutions was directly dropped onto the flowers of four-week-old Arabidopsis plants, respectively. Control plants were treated with distilled water solutions that contained the same concentration of Tween-20. After one to two days, phenotypes of the treated flowers were investigated.Sucrose and trehalose were respectively dissolved in water for preparation of 50 mM working solutions containing 0.05% . The supernatants were collected and decolorized by adding 2 mg of Reagent Five at 80 °C to 1.0 mL of extract solution, incubating for 30 min. After centrifugation , the supernatants were collected for sucrose determination using a NanoDrop 2000 spectrophotometer .For the extraction of sucrose compounds, the inflorescences were harvested from four-week-old g, China ). Approxp < 0.05, ** p < 0.01).Results are expressed as the means ± SE of data. The relative expression level of genes was obtained from at least three biological replicates for each sample as above described. Data for sucrose determination experiment were obtained from at least three independent experiments. Data for days and rosette leaves were obtained from 16 independent plants. One-way ANOVA was used to compare the differences between means of samples and to determine the statistical significance  method [JcTTPJ in Jatropha flower development.The 9 system  or knock) method , will be


The identification of glycoside hydrolases (GHs) for efficient polysaccharide deconstruction is essential for the development of biofuels. Here, we investigate the potential of sequential HMM-profile identification for the rapid and precise identification of the multi-domain architecture of GHs from various datasets. First, as a validation, we successfully reannotated >98% of the biochemically characterized enzymes listed on the CAZy database. Next, we analyzed the 43 million non-redundant sequences from the M5nr data and identified 322,068 unique GHs. Finally, we searched 129 assembled metagenomes retrieved from MG-RAST for environmental GHs and identified 160,790 additional enzymes. Although most identified sequences corresponded to single domain enzymes, many contained several domains, including known accessory domains and some domains never identified in association with GH. Several sequences displayed multiple catalytic domains and few of these potential multi-activity proteins combined potentially synergistic domains. Finally, we produced and confirmed the biochemical activities of a GH5-GH10 cellulase-xylanase and a GH11-CE4 xylanase-esterase. Globally, this “gene to enzyme pipeline” provides a rationale for mining large datasets in order to identify new catalysts combining unique properties for the efficient deconstruction of polysaccharides. GHs are found as single domain proteins (SDGHs) or associated with accessory domains such as carbohydrate binding modules (CBMs) within multi-domain GHs (MDGHs)2. In MDGHs, CBMs enhance enzyme-substrate interaction by anchoring the catalytic domain to the substrate3. The anchoring reduces diffusion from the substrate and locally increases the concentration of catalytic domains4, thus improving the overall polysaccharide degradation3.Glycoside Hydrolases (GHs) are Carbohydrate-Active Enzymes (CAZy) that catalyze the hydrolysis of the glycosidic linkage in polysaccharides  and oligosaccharides 6, whereas the breakdown of chitin, from arthropods and fungi, is important in both marine8 and terrestrial ecosystems12. Next, in the gut of animals, microbial GHs target polysaccharides, supplement the lack of endogenous enzymes14 and thus contribute to the processing of complex carbohydrates during digestion18. Finally, GHs are essential for the biofuel industry, as plant based polysaccharides constitute a major source of sustainable and renewable material capable of providing liquid transportation fuel22.GHs support essential processes for ecosystem function and for biotechnology. Among others, in land ecosystems, the deconstruction of plant biomass by microbial GHs is essential24 and bioinformatic annotation systems25. The precise identification of GH-genes and proteins is essential in order to understand how microbes support key functions across ecosystems26 and to identify new enzymes for biotechnological application27.Many GH-genes and proteins have been identified in a growing number of sequenced genomes and environmental samples thanks to the use of activity-driven screening28 combined with publicly accessible HMM-profiles from the PFam database29, here referred to as the GeneHunt approach30, to detect GH-sequences and investigate their detailed architecture 2. More precisely, we first validated the GeneHunt approach by re-annotating the biochemically characterized GHs listed on the CAZy database (as of June 2018)1. As described for cellulases, xylanases, and chitinases30, we expected the PFam-based annotation to correctly identify most of the proteins from the major GH families, although rare and recently identified GH-families would display inconsistencies. Next, we identified GHs in the M5nr database (version 13.12.15) containing 43,098,145 non-redundant, mostly microbial, protein sequences31. This collection of sequences derived from the major sequence database serves as the reference database for the MG-RAST annotation pipeline32. Finally, we identified the detailed multi-domain architecture of GH proteins in assembled, publicly accessible, metagenomes from MG-RAST. We hypothesized that, across database, GH proteins would exist primarily as single domain enzymes with low frequency of MDGHs as identified in sequenced microbial genomes10. We also expected that identified MDGHs would mostly consist in association between GH domains and CBMs as identified in the CAZy database1. Among the MDGHs, we expected to identify proteins with multiple catalytic domains and identified as potential multi-activity GHs (MAGHs). These MAGHs would display multiple catalytic domains with potential synergistic activities. In these proteins, synergistic interactions between catalytic domains would result from the complementarity of the associated domains and from a proximity effect3. More precisely, we envisioned (i) parallel pathway synergy where the combined catalytic domains target distinct, yet physically associated, substrates  and (ii) debranching synergy where one catalytic domain cleaves the side groups in substituted polysaccharides or cleaves branch points in reticulated polysaccharides, thus increasing the accessibility of the polysaccharide backbone for the second catalytic domain . Relative to SDGH and MDGH (with CBMs), these MAGHs represent vastly untapped enzymatic diversity with great potential for improved biomass deconstruction35.In order to identify new catalysts for biomass degradation, we examined the performance of sequential Hidden Markov Model (HMM) identificationsGlobally, this work provides the rationale and validation for the detailed and rapid detection of most identified GH families and associated domains from a variety of datasets, ranging from biochemically-characterized enzymes, the largest non-redundant sequence database, and assembled metagenomes derived from various environments. In addition, this work provides an exhaustive list of domains associated with GH domains, explores the diversity of multi-domain and multi-activity GHs, and identifies new types of catalysts with potential for biotechnological application.Sinapis alba, CAA42536.1). Likewise, most GH families listed on the CAZy database were consistently identified using the GeneHunt approach (Table S1). Regarding GHs targeting cellulose, xylan, and chitin, the GeneHunt approach provided a systematic and consistent annotation for potential cellulases from GH5, GH6, GH7, GH8, GH9, GH12, GH44, GH48, for potential xylanases from GH10 and GH11, and for potential chitinases from GH18, GH19, and GH85. Conversely in a few GH families including GH16, GH22, GH52, and potential cellulases from GH45 and xylanases from GH30, fewer than 90% of the proteins were annotated consistently using the GeneHunt approach. Among the GH families that could not be identified were some families that have been reclassified  and some GH families with reduced number of sequences. Having no specific HMM profile, members of these families were eventually assigned to other GH families . Finally, some GH families were associated with several Pfam IDs corresponding to various subdomains  such as GH30, GH36, GH49, and GH79. Globally, the sequences from these GH families with questionable Pfam-based annotation accounted for <3% of the analyzed sequences.First, in order to evaluate the GeneHunt approach, we (re)annotated the sequences of biochemically characterized GHs listed on the CAZy database. GeneHunt consistently annotated 7,620 GH sequences, out of 7,920 tested proteins . More precisely, >90% of the identified proteins with a domain from GH families 1, 7, and 34 were SDGHs Fig. , whereasPF00754 domains associated with GH20, GH2, GH5, GH13, GH43, and GH30 and 1,013 FIVARPF07554 domains associated with GH85, GH20, GH31, GH43 and GH13 whereas 698 BIG_2PF02368 domains were associated with GH32, GH3, GH13, GH42, GH43, and GH10 among others.Associated with these GH domains, we found many potential non-GH CAZy domains including lipases  and polysaccharide deacetylases  listed as carbohydrate esterase (CEs) in the CAZy database Fig. . We alsoPF00089, LPMO_10PF03067, B-lectinPF01453) whereas 401 CBM3PF00942, 307 Dockerin-1PF00404, 187 CBM2PF00553, 1,305 CelD_NPF02927, and 89 fibronectin-3PF00041 were the most abundant domains associated with GH9, sometimes in complex associations  and several catalytic domains including GH6 (n = 6), GH9 (n = 3), GH10 (n = 4), GH11 (n = 11), GH12 (n = 16), GH18 (n = 12), and GH44 (n = 5)  were associated to CBM2PF00553 or CBM10PF02013. Finally, the 2,117 identified GH8, were found in none of the previously identified association, formed no multi-GH8, and clustered with potential chitinolytic domains .In order to further investigate the diversity of MDGHs with potential for cellulose, xylan, and chitin deconstruction we next investigated the domain-co-occurrence network and focused on associations observed more than once Fig. . Regardi 5) Figs  and 3A. PF00734 and CBM4_9PF02018) and DockerinPF00404 domains for cellulosome assembly  and many other domains such Lipase GDSL-2PF1347, Ricin B lectin 2PF1420, and several domains with unknown function . In addition, several of these potential chitinases contained CBMs such as CBM14PF01607 (n = 254), CBM5/12PF02839 (n = 10), and CBM2PF00553 (n = 32). Finally, among the 1,163 listed GH85 domains, 7 multi-GH85s were identified. We also identified 221 Big-3PF07523, 227 F5_F8_type_CPF00754, and 176 FIVARPF07554 associated to GH85, among others , and many catalytic  and non-catalytic domains  not listed on the CAZy database.Next, we used the GeneHunt approach to identify GH domains in 129 publicly accessible assembled metagenomes from MG-RAST , 9 GH7s, 746 GH8s (1 multiGH8), 71 GH12s, 132 GH44s, 17 GH45s (1 multiGH45), and 36 GH48s (4 multiGH48s). Noteworthy, beside potential cellulases with repeated domains, 97 MDGH cellulases displayed at least 2 potential catalytic domains targeting cellulose  Figs  and 4B. Finally, chitinase domains including 3,286 GH18s (25 multiGH18s), 627 GH19s, and 247 GH85s (1 mGH85) corresponded to 3,888 proteins including 2,638 SDGHs and 1,250 MDGHs. Interestingly, 26 multi-domain chitinases displayed 2 potential chitinolytic domain.Beside these multi-domain cellulases, xylanases, and chitinases we identified several potential cellulase-xylanase , cellulase-chitinase , xylanase-chitinase , and several other potential multi-activity enzymes  and a few contained several catalytic domains. These corresponded to potential multi-activity proteins  with repeated domains such as proteins with multiple GH18 or multiple GH5 domains or proteins with distinct catalytic domains combined together Fig. . AlthougE. coli.Based on the domain associations and knowing the function of several GH families, some identified MAGHs potentially displayed interesting synergies among the catalytic domains. Thus, in order to further demonstrate the biotechnological potential of these MAGHs we selected two unique proteins identified in distinct environmental datasets for sequence optimization and gene synthesis to proceed with heterologous protein production in 36, consisting of a potential cellulase from GH5 associated with a potential xylanase from GH10  is a 377 amino acid protein, identified in a marine metagenomeH10 Fig. . As expeCMC Fig. .37, contained a potential GH10-xylanase associated with a domain identified as a potential polysaccharide deacetylase and 2 CBM4/9sPF02018 , derived from a human gut sample018 Fig. .29, can be used to identify the vast majority of characterized GHs (>98%) listed on the CAZy database1. The identification of rare or newly defined GH families such as the GH family 156 (introduced in October 2018)1 with just 7 identified sequences, having no HMM-profile available, is not yet possible using this approach30. For these families, until more sequences are identified and HMM-profiles created, similarity searches  using a custom database is an alternative , the GeneHunt approach performs two sequential HMM-profile identifications. The first search, for all the protein sequences, is performed using a small custom HMM-profile database whereas the second scan, only for the potential positive hits derived from the first step, uses the entire Pfam A database29. This approach identifies all the domains associated with GHs including the ones not listed in the custom database while minimizing the number of sequence analyses, and thus is faster than a direct scan against the complete Pfam A database. This approach can be adjusted at will by searching new HMM-profiles in the small custom database, including HMM-profiles derived from dbCAN and other domains of interest such a susD-transportersPF12741 or lipasesPF00151. In this context, the GeneHunt approach allows the identification and investigation of GH architecture in large databases such as sequenced bacterial genomes from the PATRIC database40, fungal genome from the Mycocosm database41 and MG-RAST32, as described here.The GeneHunt approach, using publicly accessible HMM-profiles from the Pfam A database1), in a large non-redundant database (M5nr), and in 129 environmental datasets from MG-RAST32, we identified hundreds of accessory non-catalytic and catalytic domains associated with GH domains. Most identified MDGHs consisted of a GH domain associated with some non-catalytic accessory domain . Beside many well-characterized domains for non-GH CAZymes and carbohydrate binding modules (CBM)1, hundreds of domains are associated with GHs. Among others, the FIVARPF07554 domain, found in various architectures, binds fibronectin and is sometimes linked to methicillin resistance43, whereas the F5/F8 type CPF00754 binds phospholipid on the surface of endothelial cells and adheres to glycoprotein in bovine milk45. The BIG_2 domain has been shown to be involved in cell-adhesion46. The systematic association of these various domains with GH domains provides insights on the modular nature of GHs in microbes. It has been shown that GHs can have multiple non-catalytic domains such as SusD-like domainsPF1274147 or CelD_NPF02927 domains48, which mediate xyloglucan-binding for cellular intake or cellulose binding by certain cellulases respectively. These non-catalytic domains play a major role in substrate binding, and thus potentially, affect the overall catalytic efficiency of associated catalytic domains. The frequent association of poorly characterized domains (e.g. domains of unknown function - DUF) could be used to infer and test potential domain activities. Based on their frequency one could identify poorly characterized domains  systematically associated with specific GH domains to infer and test their function.In addition to identifying 483,000 sequences for GHs in a curated database , parallel pathway synergy (PPS), and debranching synergy (DS). In LPS the first catalytic domain is expected to release the substrate of the second catalytic domain whereas in PPS  the catalytic domains target distinct yet physically associated substrates. Finally, in DS the first catalytic domain cleaves the side groups in substituted polysaccharide thus increasing the accessibility of the polysaccharide backbone . Although rare, these MAGHs with multiple catalytic domains represent potential robust hydrolytic systems with reduced inhibition by the product54 and display proximity effect analogous to carbohydrate binding modules3. In addition, MDGHs with DS and including some esterase activity, could possibly disrupt the xylan-lignin complex and thus improve the xylan deconstruction by associated xylanases56. Finally, depending on the processivity  of individual catalytic domain, some MAGHs could display unique modes of action35.Identifying the complete set of domain combinations in MDGHs and MAGHs is a prerequisite to investigate the evolution of the protein domains from simple to complex multi-domain enzymeFinally, MAGHs combining distinct catalytic domains, while being encoded by single genes, can easily be edited , cloned, and expressed in various hosts. In this context, the ever-growing number of accessible sequences-datasets  provides an unprecedented opportunity to identify new biotechnologically interesting catalysts. In addition, investigating the domain association in nature also highlights new ways to associate protein domains in order to take advantage of nature diversity for the purpose of synthetic biology.2. More precisely, GeneHunt uses sequential HMM-profile searches28 to rapidly identify the detailed multi-domain organization of proteins containing a domain of interest . First, selected HMM-profiles for domains of interest (Table S1) derived from the Pfam-A database29 are searched (HMMsearch) in protein datasets. Then, the protein sequences of the potential positive hits are scanned against the entire Pfam-A database (HMMscan). The first search is fast and inaccurate, whereas the second scan identifies all the domains, not just the domains of interest, and removes the false positive hits. Although relatively slow, this second scan is performed on narrowed sets of sequences, thus making the overall process faster than a direct comparison of the entire dataset using the entire Pfam-A database. GeneHunt is publicly accessible on https://github.com/renober/GeneHunt_V1.Briefly, the GeneHunt approach provides a Pfam-based domain-specific annotation of protein sequences1, as of June 2018 (Supplementary Data). Next, we retrieved and reannotated the M5nr database  containing ~43 × 106 mostly microbial non-redundant protein sequences31. Finally, we retrieved the protein sequences from 129 publicly accessible assembled metagenomes from MG-RAST (Table S2) using the MG-RAST’s “application programming interface” (API)57.To test the GeneHunt approach, we first manually retrieved and reannotated 7,920 sequences for biochemically characterized GHs listed on the CAZy databaseE. coli, and cloned in the pET151 in order to incorporate the pelB signal peptide in the N-terminal end and a His-tag at the C-terminal end of the proteins . The plasmids were then introduced into competent E. coli BL21(DE3) . Heterologous protein expression was carried out in Lysogenic Broth at 37 °C for four hours by adding 0.4 mM isopropyl-D-1-thiogalactopyranoside  when the OD600nm reached ~0.5. After centrifugation, the cell pellet was resuspended in 20 mM Tris-HCl (pH 8.0) and the cells were disrupted by sonication. Proteins from the cytoplasmic fraction were recovered by centrifugation at 20,000 × g for 40 min. Then enzymatic activities were tested qualitatively using chromogenic substrates. More precisely, azurin-cross linked xylan  was used to detect xylanase, Trypan-Blue:CarboxyMethyl-Cellulose 23 was used for cellulase, and esterase activity was tested by incubating the extract with tributyrin in presence of the pH-indicator Spirit Blue58. Xylanolytic and cellulolytic activities were visualized after incubation for 4 hours at room temperature whereas tributyrin hydrolysis required a 48 hours incubation. Thus, in order to discriminate the recombinant activity from “residual activity” from the E. coli BL21(DE3) used for protein production, the cytoplasmic fraction of non-induced cells was used as negative control for tributyrin assay.The DNA sequences of two potential multi-activity GHs were first optimized for expression in 57 synchronous GET requests: http://api.metagenomics.anl.gov/m5nr/md5/SequenceID?sequence=TRUE).Data were processed using R (Version 1.1.456) and the packages ggplot2, gplots, plyr, dplyr, reshape, reshape2, and igraph. PFam-based annotation of biochemically characterized GH listed on the CAZy database are in Supplementary File 1 and include the sequence ID, the taxonomic origin, the original annotation from CAZy, the EC-classification, and the GH domains identified using GeneHunt. Detailed GH-sequences annotation derived from M5nr database is in Supplementary File 2. Sequence from the M5nr database can be retrieved directly from the MG-RAST portal using MG-RAST API57 synchronous GET requests: http://api.metagenomics.anl.gov/download/mgmid?stage=650.Detailed GH-sequences annotation derived from M5nr database is in Supplementary Data Supplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3


A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane protein that functions as a “molecular scissor” to cleave the extracellular regions from its transmembrane target proteins. ADAM10 is well characterized as the ligand-dependent activator of Notch proteins, which control cell fate decisions. Indeed, conditional knockouts of ADAM10 in mice reveal impaired B-, T-, and myeloid cell development and/or function. ADAM10 cleaves many other leukocyte-expressed substrates. On B-cells, ADAM10 cleavage of the low-affinity IgE receptor CD23 promotes allergy and asthma, cleavage of ICOS ligand impairs antibody responses, and cleavage of the BAFF–APRIL receptor transmembrane activator and CAML interactor, and BAFF receptor, reduce B-cell survival. On microglia, increased ADAM10 cleavage of a rare variant of the scavenger receptor triggering receptor expressed on myeloid cells 2 may increase susceptibility to Alzheimer’s disease. We and others recently showed that ADAM10 interacts with one of six different regulatory tetraspanin membrane proteins, which we termed the TspanC8 subgroup, comprising Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. The TspanC8s are required for ADAM10 exit from the endoplasmic reticulum, and emerging evidence suggests that they dictate ADAM10 subcellular localization and substrate specificity. Therefore, we propose that ADAM10 should not be regarded as a single scissor, but as six different scissors with distinct substrate specificities, depending on the associated TspanC8. In this review, we collate recent transcriptomic data to present the TspanC8 repertoires of leukocytes, and we discuss the potential role of the six TspanC8/ADAM10 scissors in leukocyte development and function. Shedding can initiate intracellular signal transduction emokines . The ADAactivity .One of the best-characterized ADAMs is ADAM10, due to its essential role in ligand-dependent cleavage of Notch proteins to initiate Notch signaling . Indeed,The tetraspanins are a superfamily of transmembrane proteins expressed in multicellular eukaryotes. They are characterized by four transmembrane domains and small and large extracellular loops Figure , the latDrosophila  of T-cells in an ammation . The mecammation , nor on ammation . Among tammation , 51. Howammation . Consistammation . HoweverAn important role for ADAM10 in B-cell development and function has been demonstrated using B-cell-specific ADAM10-knockout mice, made by crossing ADAM10 floxed mice with CD19-Cre mice . Early dB-cell-specific ADAM10-knockout mice have a striking reduction in antibody responses following immunization, associated with impaired germinal center formation in secondary lymphoid tissues . The mecWith the possible exception of Notch2, the best-studied ADAM10 substrate on B-cells is CD23, the low-affinity IgE receptor, which regulates allergic and inflammatory responses , 15. IndADAM10 is emerging as a regulator of the BAFF–APRIL system which controls B-cell homeostasis. The two ligands are BAFF and APRIL, while the three receptors are B-cell maturation antigen, TACI, and BAFFR, the latter of which binds BAFF but not APRIL. ADAM10 can shed TACI to release a soluble ectodomain that acts as a decoy receptor, binding to BAFF and APRIL and so inhibiting survival of B-cells . In addiIn human B-cells, Tspan33 expression is highest, followed by Tspan14, Tspan17, and then Tspan5 Figure A. In mouDendritic cell-specific ADAM10-knockout mice have been generated by crossing ADAM10 floxed mice with CD11c-Cre mice . These mSpecific knockout of ADAM10 in myeloid cells has been investigated by crossing ADAM10 floxed mice with LysM-cre mice . This doAn additional study generated mice with leukocyte- and myeloid-specific ADAM10 deficiency by crossing floxed ADAM10 mice with Vav-Cre and LysM-Cre mice, respectively. In an inflammatory lung model, neutrophil and monocyte recruitment are reduced by approximately 50% in the absence of ADAM10 . The undFinally, the scavenger receptor TREM2 has been recently identified as an ADAM10 substrate with a potential role in Alzheimer’s disease , 20. TREThe six scissor hypothesis suggests that ADAM10 should be studied in the context of its regulatory tetraspanins. In leukocytes, the relatively high expression of Tspan14, together with its capacity to promote Notch activation, suggest that the Tspan14/ADAM10 complex may be critical for leukocyte development and function. The future analyses of cells and mice deficient in Tspan14, and other TspanC8s, will determine which scissor cleaves which substrates. This may direct therapeutic targeting of individual TspanC8/ADAM10 complexes, using antibodies or small molecules, to modulate a specific substrate while avoiding the toxicity of global ADAM10 targeting. Such an approach could provide new treatments for ADAM10-associated diseases, including T-ALL, asthma, atherosclerosis, and Alzheimer’s disease.AM, CK, JS, NH, and MT: conception, design, and writing of the manuscript. AK: data analysis and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.