Digesting gluten with oral endopeptidases to improve the management of celiac disease

In our editorial,we want to comment on the article by Stefanolo et al titled“Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet”.Celiac disease is an immune-mediated disorder triggered by dietary gluten in genetically predisposed individuals.Although avoiding gluten can permit patients to live symptom-free,ongoing voluntary or involuntary exposure to gluten is common and associated with persistent villous atrophy in small bowel mucosa.As villous atrophy predisposes patients to life threatening complications,such as osteoporotic fractures or malignancies,therapeutic adjuncts to gluten-free diet become important to improve patients’quality of life and,if these adjuncts can be shown to improve villous atrophy,avoid complications.Oral administration of enzyme preparations,such as endopeptidases that digest gluten and mitigate its antigenicity to trigger inflam-mation,is one clinical strategy under investigation.The article is about the utility of one endopeptidase isolated from Aspergillus niger.We critique findings of this clinical trial and also summarize endopeptidase-based as well as other strategies and how they can complement gluten-free diet in the management of celiac disease.

Relationship Between Endopeptidase and H_2O_2 During the Aging of Wheat Leaf

The relationship between hydrogen peroxide (H 2O 2) and endopeptidase(EP) in wheat ( Triticum aestivum L. cv. Yanmai 158) leaves was studied during natural and artificial aging. Rapid accumulation of endogenous H 2O 2 and marked increase of EP activity were observed during the later phase of aging. A new EP isozyme with higher activity was detected by electrophoresis on polyacrylamide gels containing denatured heamoglobin. With the increase of exogenous H 2O 2, the activity of EP increased at first and then decreased.

Senescence-Related Endopeptidase Isozymes in Spirodela polyrrhiza Half-fronds Detected by Gel Electrophoresis

[Objective]To study endopeptidases in Spirodela polyrrhiza half-fronds during senescence and their characters. [Method]Changes in endopeptidase isoenzymes of the Spirodela polyrrhiza half-fronds during senescence were detected by gelatin-SDS-PAGE electrophoresis,and their types were analyzed with protease inhibitors. [Result]Six endopeptidases were detected in the Spirodela polyrrhiza half-fronds during senescence. Among them,HEP1,HEP2,HEP4 and HEP6 （high molecular-weight endoprotease） were senescence-related endopeptidases. [Conclusion]The metalloendopeptidase plays significant roles at the early stage of senescence,and the cysteine endopeptidase are the most abundant at the late stage of senescence.

Characterization of Endopeptidases in Wheat Leaves During Dark-induced Senescence

The characterization of senescence-associated endopeptidase (EP) isoenzymes in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was performed. It was found that there was much higher endoproteolytic activity in dark-induced wheat leaves than in control. Six endopeptidase isoenzymes (EP1-EP6) were identified by natural gradient-polyacrylamide gel electrophoresis (PAGE) co-polymerized gelatin in the gel, five of which (EP1, EP2, EP4, EP5 and EP6) were only detected in senescing leaves. Treatment with 6-benzyl aminopurine (6-BA) delayed the expression of these EP isoenzymes and abscisic acid (ABA) accelerated it. The activity of EP3 could be detected at a wider range of pH and temperature levels while EP4, EP5 and EP 6 could be only detected at pH 4-5 and 30 -45 degreesC, EP1 and EP2 at pH 3-5 and 30-45 degreesC. All of the EP isoenzymes showed high thermal stability, especially EP3, EP5 and EP6 which still had activitiy even by incubation at 55 degreesC for 1 h. By using different class-specific inhibitors, EP1 and EP2 were characterized as metal-dependent cysteine-proteases, EP4 as a serine-protease.

Changes in Endopeptidase Activity during Photosynthetic Declination in Rice Leaf

Two japonica rice varieties, Wuyujing 3 and 97-7, were used to study the changes in contents of soluble protein, free amino acids and endopeptidase activity during photosynthetic declination. The content of soluble protein in flag leaf of cv. Wuyujing 3 was higher than that of cv. 97-7, but decreased rapidly in Wuyujing 3. Free amino acids in flag leaf and the thirteenth leaf of Wuyujing 3 started to increase 10 days before the turning point of photosynthetic declination (TPPD), while it occurred just 1-2 days before TPPD in the flag leaf and the thirteenth leaf of 97-7. During reversible phase of photosynthetic declination, endopeptidase activity remained at a low level and increased slightly only in the later part of this phase. Then it rose up rapidly at irreversible decline phase and reached a very high level. For Wuyujing 3, the change in endopeptidase activity in the thirteenth leaf was parallel to that in flag leaf. However, for 97-7, the rapid increase of endopeptidase activity in the thirteenth leaf started later than that of flag leaf. The results implied that the rate of protein breakdown and conversion to transportable nitrogen in leaves of 97-7 was slower than that in leaves of Wuyujing 3 during photosynthetic declination and it led to relatively lower seed setting rate and fully filling grains rate in 97-7. This may be one of the important reasons why 97-7 could not bring the high yield potentiality into play and the findings may be taken into consideration while breeding for high potential varieties in future.

Peptidase Activities of Tripeptidyl Peptidase Ⅰ (TPP Ⅰ): Exopeptidase and Endopeptidase

The defect of TPP Ⅰ causes a disease, late infantile neuronal ceroid lipofuscinosis（LINCL, CLN2）. To investigate the bio-activity of tripeptidyl peptidase Ⅰ （TPP Ⅰ ） from rat kidneys, the effects of digestion of angiotensin Ⅱ （Ang Ⅱ ） and a synthetic endo-type substrate（ Gly^1-Lys-Pro^5-lie-Pro^5-Phe-Phe-Arg-Leu-Lys^10） via TPP I were ana- lyzed by HPLC and TOF-MS. The data suggest that the degradation rate of Ang I1 can reach 18. 2% by the rat TPP I and DRV（Asp-Arg-Val） can be released from N-termini of Ang Ⅱ within 16 h. In addition, the synthetic endotype substrate is cleaved at the same position between Phe6 and Phe^7. Accordingly, TPP Ⅰ shows two kinds of peptidase activities. One is a tripeptidyl peptidase activity and the other is a pepstatin insensitive carboxyl endopeptidase activity. Tripeptidyl peptidase activity and pepstatin insensitive carboxyl endopeptidase activity seem to be dual phases of one enzyme, TPP Ⅰ.

Therapeutic Effect of Prolyl Endopeptidase Inhibitor in High-fat Diet-induced Metabolic Dysfunction-associated Fatty Liver Disease

Background and Aims:Prolyl endopeptidase(PREP)is a serine endopeptidase that participates in many pathological processes including inflammation,oxidative stress,and autophagy.Our previous studies found that PREP knockout exhibited multiple benefits in high-fat diet(HFD)or methionine choline-deficient diet-induced metabolic dysfunctionassociated fatty liver disease(MAFLD).However,cumulative studies have suggested that PREP performs complex functions during disease development.Therefore,further understanding the role of PREP in MAFLD development is the foundation of PREP intervention.Methods:In this study,an HFD-induced MAFLD model at different time points(4,8,12,and 16 weeks)was used to explore dynamic changes in the PREP proline-glycine-proline(PGP)/N-acetyl-seryl-aspartyllysyl-proline(AcSDKP)system.To explore its potential value in MAFLD treatment,saline,or the PREP inhibitor,KYP-2047,was administered to HFD-induced MAFLD mice from the 10th to 16th weeks.Results:PREP activity and expression were increased in HFD-mice compared with control mice from the 12th week onwards,and increased PREP mainly resulted in the activation of the matrix metalloproteinase 8/9(MMP8/9)-PREP-PGP axis rather than the thymosin B4-meprin a/PREP-AcSDKP axis.In addition,KYP-2047 reduced HFD-induced liver injury and oxidative stress,improved lipid metabolism through the suppression of lipogenic genes and the induction of B-oxidation-related genes,and attenuated hepatic inflam-mation by decreasing MMP8/9 and PGP.Moreover,KYP2047 restored HFD-induced impaired autophagy and this was veri-fied in HepG2 cells.Conclusions:These findings suggest that increased PREP activity/expression during MAFLD de-velopment might be a key factor in the transition from sim-ple steatosis to steatohepatitis,and KYP-2047 might possess therapeutic potential for MAFLD treatment.

Characters of Cysteine Endopeptidases in Wheat Endosperm during Seed Germination and Subsequent Seedling Growth

The endopeptidases （EPs） in wheat endosperm during seed germination and subsequent seedling growth were characterized by gradient-polyacrylamide gel electrophoresis with gelatin copolymerized into the gel. Four cysteine EPs （EP1, EP2, EP3 and EP4） were detected in wheat endosperm during the 7 d growth after seed imbibition. The results also showed that the activities of all of these EPs increased continuously, and EP2 first appeared and had the highest proteolytic activity among the four EPs in this experimental process. The optimum pH and temperature of all four EPs were 4.0 and 40.0 ~C. All EPs were completely inhibited by 25 μmol/L E-64 and had no good thermal stabilities, especially EP1. In addition, these EPs had different substrate specificities to albumins, globulins, gliadins and glutenins; the main storage proteins of mature wheat endosperm. Among them, EP2 had the highest proteolytic activities on globulins, gliadins and glutenins, and might be the most important and specific EP with potential to be tightly correlated with seedling development.

Endopeptidase Isoenzyme Characteristics in Cucumis sativus Leaves During Dark-induced Senescence

The changes and characteristics of endopeptidase （EP） isoenzymes in cucumber （Cucumis sativus L.） leaves during dark-induced senescence were investigated by activity staining after gradient-polyacrylamide gel electrophoresis （G-PAGE） containing co-polymerized gelatin as substrate. The results showed that both the chlorophyll and the protein contents of leaves were decreased, and the protein degradation was correlated with the increase of proteolytic activity during the course of leaf senescence. Meanwhile, nine cucumber endopeptidases isoenzymes （CEP） with 140, 120, 106, 94, 76, 55, 46, 39 and 35 kDa molecular weights were detected. Four of these, CEP2, 3, 4 and CEP9 appeared all the time, but the changes of the activity were different during incubation. Another four CEPs （CEP5, 6, 7 and CEP8） whose activities increased with dark-induced time were only detected in senescent leaves. Furthermore, the biochemical properties of these nine CEP were also characterized. All the CEPs had high activities from 35 ℃ to 45 ℃, and the optimum temperature was found to be 40 ℃. However, the activities of CEPs were not detected below 25 ℃ or over 60 ℃. The activity bands appeared at a wide range of pH from 5.0 to 9.0, but the optimum pH was found at 7.0. No CEPs were detected at pH 4 or pH 10. By inhibition analysis we concluded that CEP2, 3, 4 and CEP9 were serine endopeptidases and CEP6 was a kind of cysteine protease. It is suggested that serine endopeptidases might play a major role in cucumber leaf senescence, and for the first time, six senescencerelated endopeptidases （CEP1, 5, 6, 7, 8 and 9） were found in cucumber leaves.

Pharmacological inhibition of asparaqinyl endopeptidase by δ-secretase inhibitor 11 mitigates Alzheimer's disease-related pathologies in a senescence-accelerated mouse model

Background:Currently,there is no cure for Alzheimer's disease(AD).Therapeutics that can modify the early stage of AD are urgently needed.Recent studies have shown that the pathogenesis of AD is closely regulated by an endo/lysosomal asparaginyl endopeptidase(AEP).Inhibition of AEP has been reported to prevent neural degeneration in transgenic mouse models of AD.However,more than 90% of AD cases are age-related sporadic AD rather than hereditary AD.The therapeutic efficacy of AEP inhibition in ageing-associated sporadic AD remains unknown.Methods:The senescence-accelerated mouse prone 8(SAMP8)was chosen as an approximate model of sporadic AD and treated with a selective AEP inhibitor,δ-secretase inhibitor 11.Activation of AEP was determined by enzymatic activity assay.Concentration of soluble amyloid β(Aβ)in the brain was determined by ELISA.Morris water maze test was performed to assess the learning and memory-related cognitive ability.Pathological changes in the brain were explored by morphological and western blot analyses.Results:The enzymatic activity of AEP in the SAMP8 mouse brain was significantly higher than that in the agematched SAMR1 mice.The half maximal inhibitory concentration(IC_(50))for δ-secretase inhibitor 11 to inhibit AEP in vitro was around 150 nM.Chronic treatment with δ-secretase inhibitor 11 markedly decreased the brain AEP activity,reduced the generation of Aβ_(1-40/42) and ameliorated memory loss.The inhibition of AEP with this reagent not only reduced the AEP-cleaved tau fragments and tau hyperphosphorylation,but also attenuated neuroinflammation in the form of microglial activation.Moreover,treatment with 6-secretase inhibitor 11 prevented the synaptic loss and alleviated dendritic disruption in SAMP8 mouse brain.Conclusions:Pharmacological inhibition of AEP can intervene and prevent AD-like pathological progress in the model of sporadic AD.The up-regulated AEP in the brain could be a promising target for early treatment of AD.The δ-secretase inhibitor 11 can be used as a lead compound for translational development of AD treatment.

Activity Gel Analysis of Endopeptidases in Rose Petals

Poor resolution for the identification of endopeptidase(EP) activity in activity gel assays is a critical issue in the analysis of the postharvest physiology of rose petals. In this paper, major factors influencing EP activity gel assays were evaluated. The results showed that a phosphate(NaH_2PO_4/Na_2HPO_4) buffer favors the detection of clear EP bands, as compared to Tris–HCl buffer. Removal of salts and pigments with Sephadex G-25 columns was vital to the measurement of EP activity in rose petal extracts. For optimal resolution of bands, we show that before electrophoresis,samples should be treated for 10 min at 40 °C. Additionally, electrophoresis should be done in 12% SDS–PAGE co-polymerized with 0.15%(w/v) gelatin. After electrophoresis, the optimal incubation temperature and pH are 42 °C and 7.0, respectively. Using our optimized assay, Rh-EP1,Rh-EP2, Rh-EP3, three proteases with molecular masses of 200, 123.5, and 97.4 kD, respectively, were detected in rose petals. Experiments using EP class-specific inhibitors revealed that Rh-EP2 and Rh-EP3 were both serine proteases, while Rh-EP1 was a metalloprotease. In this study, we also measured changes in EP activity during flower opening, senescence, and water deficit stress(WDS) using our optimized activity gel assay,and found that Rh-EP3 may be more relevant to senescence in roses compared with Rh-EP1 or Rh-EP2. Changes occurring to EPs after WDS were similar to those during the period from flower opening to senescence, and Rh-EP3 activities were greatly increased by WDS treatment. Collectively, our results suggest that significant increases in Rh-EPs activities, especially increases in Rh-EP3 activity, may contribute to the flower senescence induced under WDS treatment.

Cellular response toβ-amyloid neurotoxicity in Alzheimer's disease and implications in new therapeutics

β-Amyloid(Aβ)is a specific pathological hallmark of Alzheimer's disease(AD).Because of its neurotoxicity,AD patients exhibit multiple brain dysfunctions.Disease-modifying therapy(DMT)is the central concept in the development of AD thera-peutics today,and most DMT drugs that are currently in clinical trials are anti-Aβdrugs,such as aducanumab and lecanemab.Therefore,understanding Aβ's neurotoxic mechanism is crucial for Aβ-targeted drug development.Despite its total length of only a few dozen amino acids,Aβis incredibly diverse.In addition to the well-known Aβ_(1-42),N-terminally truncated,glutaminyl cyclase(QC)catalyzed,and pyroglutamate-modified Aβ(pEAβ)is also highly amyloidogenic and far more cytotoxic.The extracel-lular monomeric Aβ_(x-42)(x=1-11)initiates the aggregation to form fibrils and plaques and causes many abnormal cellular responses through cell membrane receptors and receptor-coupled signal pathways.These signal cascades further influence many cel-lular metabolism-related processes,such as gene expression,cell cycle,and cell fate,and ultimately cause severe neural cell damage.However,endogenous cellular anti-Aβdefense processes always accompany the Aβ-induced microenvironment alterations.Aβ-cleaving endopeptidases,Aβ-degrading ubiquitin-proteasome system(UPS),and Aβ-engulfing glial cell immune responses are all essential self-defense mechanisms that we can leverage to develop new drugs.This review discusses some of the most recent advances in understanding Aβ-centric AD mechanisms and suggests prospects for promising anti-Aβstrategies.

半胱氨酸蛋白酶激动剂、X连锁凋亡抑制蛋白、半胱氨酸天冬氨酸蛋白酶3在鼻腔鼻窦鳞状细胞癌中的表达

现代肿瘤学认为肿瘤的发病与细胞凋亡数量的减少密切相关,第二个线粒体衍生的半胱氨酸蛋白酶激动剂（second mitochondria-derived activator of caspase,Smac）、X连锁凋亡抑制蛋白（X-linked inhibitor of apoptosis protein,XIAP）、半胱氨酸天冬氨酸蛋白酶3（cysteine containing aspartate specific protease,caspase-3）在细胞凋亡过程中起着重要的调控及效应作用。本研究采用免疫组化方法观察三者在鼻腔鼻窦鳞状细胞癌（nasal squamous cellcarcinoma,NSCC）及不同病理分期中的表达情况。

The Effect of Lumbrokinase on P-selectin and E-selectin in Cerebral Ischemia Model of Rat

Purpose: To find the effect of lumbrokinase (LK) on P-selectin and E-selectin in ischemic rats. Methods: Male healthy Spragur-Dawley rats weighing 180-220 g (n=90) were divided into 4 groups: (1) normal control group (n=5), (2) sham-operated group (n=35), (3) ischemic group (n=35), (4) LK group (n=15). LK 10mg/kg (2000UK activity of LK) was given by intraperitoneal injection in the LK group 30 minutes before experiment. Same volume of normal saline was given in the sham-operated group and ischemic group. The ischemic model was made by modified Haruo Nagasawa's method. Immunohistochemistry was used to observe the P-selectin and E-selectin positive cells in the ischemic region. Results: P-selectin and E-selectin positive cells in ischemic regions were observed in the ischemic group, and the peak of expression was at 6 hours and 12 hours, respectively. The similar changes were not observed in normal control group. There were only a few positive cells in the sham-operated group. In LK group, the P-selectin and E-selectin positive cells were significantly less than those in the ischemic group (P<0.05 at 3 hours after the onset, P<0.01 at 6 hours and P<0.01 at 12 hours, respectively). Conclusions: LK might significantly decrease the immunoreactions of P-selectin and E-selectin in ischemic lesion.

Early Morphological and Physiological Events Occurring During Germination of Maize Seeds

The early morphological and physiological events occurring during maize （Zea mays cv. Nongda 108） seed imbibition and germination were studied. Water uptake of seeds exhibited a triphasic pattern with a marked increase during the initial phase of imbibition, and then a slow increase, followed by a second substantial increase. Imbibition time for 10 and 50% of seed germination was about 26 and 46 h at 30℃, respectively. The relative conductivity of maize seeds dramatically decreased during the initial phase of imbibition, followed by a substantial increase. Respiratory rate of seeds gradually increased with imbibition. Length of root cap cells decreased during the initial phase and then increased; those of meristematic zone cells increased during the initial phase and then decreased; and those of elongation zone cells and of the whole elongation zone of the radicle gradually increased during germination. The contents of soluble sugars and starch in embryos gradually decreased as the activities of α- and β-amylase strikingly increased with imbibition. In the meantime, protein contents of embryos gradually decreased and free amino acid content increased. The activities of aminopeptidase and endopeptidase increased until 12 h of imbibition and then decreased. It is concluded that germination of maize seeds is mainly completed by extension of cells in the elongation zone of the radicle, and that mobilization of stored reserves in the embryo during the initial phase of imbibition is also an early event during seed germination.

Concomitant NH_4^+ Secretion During Astaxanthin Synthesis in Haematococcus pluvialis Under High Irradiance and Nitrogen Defi-cient Conditions

The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin synthesis in the cultures of high light and nitrogen-free （HF）, high light and nitrogen-repletion （HR）, and low light and nitrogen-free （LF）, （1） endopeptidase （EP） activities increased along with decrease in protein content, （2） asparagine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, （3） ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, （LR）, the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas （02 and N2）, astaxan- thin content increased as the protein level decreased. These results strongly suggest that （1） the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, （2） endopeptidase was involved in the degradative process, （3） for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.

Childhood chronic gastritis and duodenitis: Role of altered sensory neuromediators

AIM To investigate the roles of the neuropeptides vasoactive intestinal peptide(VIP), substance P(SP), and calcitonin gene-related peptide(CGRP) in chronic gastritis and duodenitis in children.METHODS Biopsy samples from the gastric and duodenal mucosa of 52 patients and 30 control subjects were obtained. Samples were taken for pathological examination, immunohistochemical staining, enzyme activity measurements and quantitative measurements of tissue peptide levels.RESULTS We observed differential effects of the disease on peptide levels, which were somewhat different from previously reported changes in chronic gastritis in adults. Specifically, SP was increased and CGRP and VIP were decreased in patients with gastritis. The changes were more prominent at sites where gastritis was severe, but significant changes were also observedin neighboring areas where gastritis was less severe. Furthermore, the degree of changes was correlated with the pathological grade of the disease. The expression of CD10, the enzyme primarily involved in SP hydrolysis, was also decreased in patients with duodenitis.CONCLUSION Based on these findings, we propose that decreased levels of VIP and CGRP and increased levels of SP contribute to pathological changes in gastric mucosa. Hence, new treatments targeting these molecules may have therapeutic and preventive effects.

C/EBPβ/AEP Signaling Drives Alzheimer's Disease Pathogenesis

Alzheimer’s disease(AD)is the most common type of dementia.Almost two-thirds of patients with AD are female.The reason for the higher susceptibility to AD onset in women is unclear.However,hormone changes during the menopausal transition are known to be associated with AD.Most recently,we reported that follicle-stimulating hormone(FSH)promotes AD pathology and enhances cognitive dysfunctions via activating the CCAAT-enhancer-binding protein(C/EBPβ)/asparagine endopeptidase(AEP)pathway.This review summarizes our current understanding of the crucial role of the C/EBPβ/AEP pathway in driving AD pathogenesis by cleaving multiple critical AD players,including APP and Tau,explaining the roles and the mechanisms of FSH in increasing the susceptibility to AD in postmenopausal females.The FSH-C/EBPβ/AEP pathway may serve as a novel therapeutic target for the treatment of AD.

Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese encephalitis virus

OBJECTIVE: To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression. METHODS: Persistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins. RESULTS: In the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed. CONCLUSIONS: The virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.

Filling the Gaps to Solve the Extensin Puzzle

Extensins （EXTs） are highly repetitive plant O-glycoproteins that require several post-translational modifi- cations （PTMs） to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then they are O-glycosylated on hydroxyproline and serine. After secretion into the apoplast, O-glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in the identification of the enzymatic machinery required to process EXTs, which includes prolyl 4-hydroxylases, glycosyltransferases, papain-type cysteine endopeptidases, and peroxidases. EXTs are abundant in plant tissues and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fastgrowing cells, although the molecular mechanisms underlying this regulation are unknown. In this review, we highlight recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in cell walls, and possible sensing mechanisms involving the Catharanthus roseus cell surface sensor receptor-like kinases located at the interface between the apoplast and the cytoplasmic side of the plasma membrane.