MYB regulates the SUMO protease SENP1 and its novel interaction partner UXT, modulating MYB target genes and the SUMO landscape.

SUMOylation is a post-translational modification frequently found on nuclear proteins, including transcription factors (TFs) and coactivators. By controlling the activity of several TFs, SUMOylation may have far-reaching effects. MYB is an example of a developmental TF subjected to SUMO-mediated regulation, through both SUMO conjugation and SUMO binding. How SUMO affects MYB target genes is unknown. Here, we explored the global effect of reduced SUMOylation of MYB on its downstream gene programs. RNA-Seq in K562 cells after MYB knockdown and rescue with mutants having an altered SUMO status revealed a number of differentially regulated genes and distinct gene ontology term enrichments. Clearly, the SUMO status of MYB both quantitatively and qualitatively affects its regulome. The transcriptome data further revealed that MYB upregulates the SUMO protease SENP1, a key enzyme that removes SUMO conjugation from SUMOylated proteins. Given this role of SENP1 in the MYB regulome, we expanded the analysis, mapped interaction partners of SENP1, and identified UXT as a novel player affecting the SUMO system by acting as a repressor of SENP1. MYB inhibits the expression of UXT suggesting that MYB is able not only to control a specific gene program directly but also indirectly by affecting the SUMO landscape through SENP1 and UXT. These findings suggest an autoactivation loop whereby MYB, through enhancing SENP1 and reducing UXT, is itself being activated by a reduced level of repressive SUMOylation. We propose that overexpressed MYB, seen in multiple cancers, may drive this autoactivation loop and contribute to oncogenic activation of MYB.

Rac-dependent feedforward autoactivation of NOX2 leads to oxidative burst.

NADPH oxidase 2 (NOX2) produces the superoxide anion radical (O2-), which has functions in both cell signaling and immune defense. NOX2 is a multimeric-protein complex consisting of several protein subunits including the GTPase Rac. NOX2 uniquely facilitates an oxidative burst, which is described by initially slow O2- production, which increases over time. The NOX2 oxidative burst is considered critical to immune defense because it enables expedited O2- production in response to infections. However, the mechanism of the initiation and progression of this oxidative burst and its implications for regulation of NOX2 have not been clarified. In this study, we show that the NOX2 oxidative burst is a result of autoactivation of NOX2 coupled with the redox function of Rac. NOX2 autoactivation begins when active Rac triggers NOX2 activation and the subsequent production of O2-, which in turn activates redox-sensitive Rac. This activated Rac further activates NOX2, amplifying the feedforward cycle and resulting in a NOX2-mediated oxidative burst. Using mutagenesis-based kinetic and cell analyses, we show that enzymatic activation of Rac is exclusively responsible for production of the active Rac trigger that initiates NOX2 autoactivation, whereas redox-mediated Rac activation is the main driving force of NOX2 autoactivation and contributes to generation of ∼98% of the active NOX2 in cells. The results of this study provide insight into the regulation of NOX2 function, which could be used to develop therapeutics to control immune responses associated with dysregulated NOX2 oxidative bursts.

Development of Noonan syndrome by deregulation of allosteric SOS autoactivation.

Ras family proteins play an essential role in several cellular functions, including growth, differentiation, and survival. The mechanism of action of Ras mutants in Costello syndrome and cancers has been identified, but the contribution of Ras mutants to Noonan syndrome, a genetic disorder that prevents normal development in various parts of the body, is unknown. Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor. In response to Ras-activating cell signaling, SOS autoinhibition is released and is followed by accelerative allosteric feedback autoactivation. Here, using mutagenesis-based kinetic and pulldown analyses, we show that Noonan syndrome Ras mutants I24N, T50I, V152G, and D153V deregulate the autoactivation of SOS to populate their active form. This previously unknown process has been linked so far only to the development of Noonan syndrome. In contrast, other Noonan syndrome Ras mutants-V14I, T58I, and G60E-populate their active form by deregulation of the previously documented Ras GTPase activities. We propose a novel mechanism responsible for the deregulation of SOS autoactivation, where I24N, T50I, V152G, and D153V Ras mutants evade SOS autoinhibition. Consequently, they are capable of forming a complex with the SOS allosteric site, thus aberrantly promoting SOS autoactivation, resulting in the population of active Ras mutants in cells. The results of this study elucidate the molecular mechanism of the Ras mutant-mediated development of Noonan syndrome.

Evolutionary expansion of polyaspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis.

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen paralogs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased the autoactivation of T7 trypsinogen threefold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side chains are preferred for maximal autoactivation, and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by NH2-terminal truncation with chymotrypsin C reduced the autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable with that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the polyaspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.NEW & NOTEWORTHY Unwanted autoactivation of the digestive protease trypsinogen can result in pancreatitis. The trypsinogen activation peptide contains a polyaspartate motif that suppresses autoactivation. This study demonstrates that evolutionary expansion of these aspartate residues in mouse cationic trypsinogen further inhibits autoactivation and enhances protection against pancreatitis.

Autoactivation of C-terminally truncated Ca2+/calmodulin-dependent protein kinase (CaMK) Iδ via CaMK kinase-independent autophosphorylation.

Ca2+/calmodulin-dependent protein kinase I isoforms (CaMKIα, ß, γ, and δ) play important roles in Ca2+ signaling in eukaryotic cells by being activated by CaMK kinase (CaMKK) through phosphorylation at a Thr residue in the activation loop. However, we have recently found that, unlike rat CaMKIα (rCaMKIα), C-terminally truncated fragments of zebrafish and mouse CaMKIδ [zCaMKIδ(1-299) and mCaMKIδ(1-297)] produced by Escherichia coli exhibit almost full activity in the absence of CaMKK. To address the CaMKK-independent activation mechanism of CaMKIδ in E. coli cells, here we performed comparative analyses between recombinant zCaMKIδ(1-299) and rCaMKIα(1-294) in vitro. By using a kinase-dead mutant of zCaMKIδ(1-299) and λ phosphatase coexpression method, we elucidated that zCaMKIδ(1-299) was highly autophosphorylated and activated in E. coli during cell culture, but rCaMKIα(1-294) was not. The major autophosphorylation site leading to activation of the kinase was Ser296, determined using mass spectrometry analysis in conjunction with site-directed mutagenesis. Furthermore, mimicking phosphorylation at Ser296 in full-length zCaMKIδ resulted in additional activation of the kinase compared with CaMKI fully activated by CaMKK. Our results provide the first evidence that CaMKIδ is activated through CaMKK-independent phosphorylation at Ser296, which might be a clue to understand the physiological regulation of CaMKIδ isoform.

A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila.

Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator.

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis.

Mutations in the PRSS1 gene encoding human cationic trypsinogen are associated with hereditary and sporadic chronic pancreatitis. High-penetrance PRSS1 mutations found in hereditary pancreatitis alter activation and/or degradation of cationic trypsinogen, thereby promoting intrapancreatic trypsinogen activation. In contrast, a number of rare PRSS1 variants identified in subjects with sporadic chronic pancreatitis cause misfolding and endoplasmic reticulum (ER) stress. Mutation p.L104P is unique among natural PRSS1 variants, since it affects the substrate binding site of trypsin. The aim of the present study was to establish the clinical significance of variant p.L104P through functional analysis. We found that p.L104P trypsin exhibited decreased activity on peptide and protein substrates; however, autoactivation was slightly accelerated. Remarkably, binding of the physiological trypsin inhibitor serine protease inhibitor Kazal type 1 (SPINK1) was decreased by 70-fold. In the presence of the trypsinogen-degrading enzyme chymotrypsin C, mutant p.L104P autoactivated to higher trypsin levels than wild-type trypsinogen. This apparent resistance to degradation was due to slower cleavage at Arg(122) rather than Leu(81) Finally, secretion of mutant p.L104P from transfected cells was markedly reduced due to intracellular retention and aggregation with concomitant elevation of ER stress markers. We conclude that PRSS1 variant p.L104P exhibits a variety of phenotypic changes that can increase risk for chronic pancreatitis. Mutation-induced misfolding and associated ER stress are the dominant effects that support a direct pathogenic role in chronic pancreatitis.

Gene conversion between cationic trypsinogen (PRSS1) and the pseudogene trypsinogen 6 (PRSS3P2) in patients with chronic pancreatitis.

Mutations of the human cationic trypsinogen gene (PRSS1) are frequently found in association with hereditary pancreatitis. The most frequent variants p.N29I and p.R122H are recognized as disease-causing mutations. Three pseudogene paralogs in the human trypsinogen family, including trypsinogen 6 (PRSS3P2), carry sequence variations in exon 3 that mimic the p.R122H mutation. In routine genetic testing of patients with chronic pancreatitis, we identified in two unrelated individuals similar gene conversion events of 24-71 nucleotides length between exon 3 of the PRSS1 (acceptor) and PRSS3P2 (donor) genes. The converted allele resulted in three nonsynonymous alterations c.343T>A (p.S115T), c.347G>C (p.R116P), and c.365_366delinsAT (p.R122H). Functional analysis of the conversion triple mutant revealed markedly increased autoactivation resulting in high and sustained trypsin activity in the presence of chymotrypsin C. This activation phenotype was identical to that of the p.R122H mutant. In addition, cellular secretion of the triple mutant from transfected HEK 293T cells was increased about twofold and this effect was attributable to mutation p.R116P. Our observations confirm and extend the notion that recombination events between members of the trypsinogen family can generate high-risk PRSS1 alleles. The pathogenic phenotype of the novel conversion is explained by a unique combination of increased trypsinogen activation and secretion.

Efficient processing of procathepsin K to the mature form.

The proteolysis of collagen fibrils by cathepsin K is a hallmark of bone catabolism and tissue degeneration. The production of active recombinant cathepsin K is central for our ability to study the mechanisms by which these processes occur. Here we report an efficient processing method for the preparation of recombinant cathepsin K expressed in Pichia pastoris. Methanol precipitation of crude media and autoactivation in the absence of a reducing agent allows for the reversible inhibition of the enzyme prior to subsequent purification steps. The resultant purified enzyme is both resistant to autolysis and effective at cleaving collagen.

Phytate-Induced Dose-Response Auto-Activation of Enzyme in Commercial Recombinant Phytase From Escherichia coli.

Background: Microbial phytase is one of the most widely used enzymes in food industries like cattle, poultry, and aquaculture food. Therefore, understanding the kinetic properties of the enzyme is very important to evaluate and predict its behavior in the digestive system of livestock. Working on phytase is one of the most challenging experiments because of some problems, including free inorganic phosphate (FIP) impurity in phytate (substrate) and interference reaction of the reagent with both phosphates (product and phytate impurity). Objective: In the present study, FIP impurity of phytate was removed, and then it was shown that the substrate (phytate) has a dual role in enzyme kinetics: substrate and activator. Material and Methods: phytate impurity was decreased by two-step recrystallization prior to the enzyme assay. The impurity removal was estimated by the ISO30024:2009 method and confirmed by Fourier-transform infrared (FTIR) spectroscopy. The kinetic behavior of phytase activity was evaluated using the purified phytate as substrate by non-Michaelis-Menten analysis, including Eadie-Hofstee, Clearance, and Hill plots. The possibility of an allosteric site on phytase was assessed by molecular docking. Results: The results showed a 97.2% decrease in FIP due to recrystallization. The phytase saturation curve had a sigmoidal appearance, and Lineweaver-Burk plot with a negative y-intercept indicated the positive homotropic effect of the substrate on the enzyme activity. A right-side concavity of Eadie-Hofstee plot confirmed it. Hill coefficient was calculated to be 2.26. Molecular docking also showed that Escherichia coli phytase molecule has another binding site for phytate very close to the active site, called "allosteric site". Conclusions: The observations strongly propose the existence of an intrinsic molecular mechanism in Escherichia coli phytase molecules to be promoted for more activity by its substrate, phytate (positive homotropic allosteric effect). In silico analysis showed that phytate binding to the allosteric site caused new substrate-mediated inter-domain interactions, which seems to lead to a more active conformation of phytase. Our results provide a strong basis for animal feed development strategies, especially in the case of poultry food and supplements, regarding a short food passage time in their gastrointestinal tract and variable concentration of phytate along with it. Additionally, the results strengthen our understanding of phytase auto-activation as well as allosteric regulation of monomeric proteins in general.

A bistable autoregulatory module in the developing embryo commits cells to binary expression fates.

Bistable autoactivation has been proposed as a mechanism for cells to adopt binary fates during embryonic development. However, it is unclear whether the autoactivating modules found within developmental gene regulatory networks are bistable, unless their parameters are quantitatively determined. Here, we combine in vivo live imaging with mathematical modeling to dissect the binary cell fate dynamics of the fruit fly pair-rule gene fushi tarazu (ftz), which is regulated by two known enhancers: the early (non-autoregulating) element and the autoregulatory element. Live imaging of transcription and protein concentration in the blastoderm revealed that binary Ftz fates are achieved as Ftz expression rapidly transitions from being dictated by the early element to the autoregulatory element. Moreover, we discovered that Ftz concentration alone is insufficient to activate the autoregulatory element, and that this element only becomes responsive to Ftz at a prescribed developmental time. Based on these observations, we developed a dynamical systems model and quantitated its kinetic parameters directly from experimental measurements. Our model demonstrated that the ftz autoregulatory module is indeed bistable and that the early element transiently establishes the content of the binary cell fate decision to which the autoregulatory module then commits. Further in silico analysis revealed that the autoregulatory element locks the Ftz fate quickly, within 35 min of exposure to the transient signal of the early element. Overall, our work confirms the widely held hypothesis that autoregulation can establish developmental fates through bistability and, most importantly, provides a framework for the quantitative dissection of cellular decision-making.

The structural basis of the multi-step allosteric activation of Aurora B kinase.

Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.

The Human Ntn-Hydrolase Superfamily: Structure, Functions and Perspectives.

N-terminal nucleophile (Ntn)-hydrolases catalyze the cleavage of amide bonds in a variety of macromolecules, including the peptide bond in proteins, the amide bond in N-linked protein glycosylation, and the amide bond linking a fatty acid to sphingosine in complex sphingolipids. Ntn-hydrolases are all sharing two common hallmarks: Firstly, the enzymes are synthesized as inactive precursors that undergo auto-proteolytic self-activation, which, as a consequence, reveals the active site nucleophile at the newly formed N-terminus. Secondly, all Ntn-hydrolases share a structural consistent αßßα-fold, notwithstanding the total lack of amino acid sequence homology. In humans, five subclasses of the Ntn-superfamily have been identified so far, comprising relevant members such as the catalytic active subunits of the proteasome or a number of lysosomal hydrolases, which are often associated with lysosomal storage diseases. This review gives an updated overview on the structural, functional, and (patho-)physiological characteristics of human Ntn-hydrolases, in particular.

Visualizing Bioabsorbable Spacer Effectiveness by Confirming the Distal-Tail of Carbon-Ion Beams: First-In-Human Report.

In particle therapy, bioabsorbable polyglycolic acid (PGA) spacer was developed to reduce the healthy organ irradiation dose, especially in the gastrointestinal tract. The PGA spacer is safe and effective; however, there are no reports that have confirmed whether the PGA spacer which inserted in the body actually stops the carbon-ion (C-ion) beams. Here, we visualized and confirmed that the PGA spacer stops the C-ion beams in the body based on the dose distribution using auto-activation positron emission tomography (AAPET). A 59-year-old dedifferentiated retroperitoneal liposarcoma patient underwent C-ion radiotherapy (C-ion RT) on referral. A month before C-ion RT initiation, the patient underwent PGA spacer placement. Postoperatively, the patient received 4.4 Gy (RBE) per fraction of C-ion RT, followed by AAPET. AAPET revealed lower positron emitter concentrations at the distal tissue ventral to the PGA spacer than in the planning target volume. In observing the efficacy of the PGA spacer, the AAPET images and the average count per second of the positron emitter suggested that the PGA spacer stopped the C-ion beams in the body in accordance with the dose distribution. Therefore, AAPET was useful in confirming the PGA spacer's effectiveness in this study, and the PGA spacer stopped the C-ion beams.

Visualisation of Range Shortening in Carbon Ion Beams and Washout of Positron Emitter: First-in-Human Report.

BACKGROUND/AIM: Auto-activation positron emission tomography (AAPET) is one of the most promising methods to verify beam range in carbon ion radiotherapy (C-ion RT). We aimed to confirm this for the first time in a clinical setting by performing AAPET in a patient with pancreatic cancer previously receiving coil embolisation of hepatic artery pseudoaneurysm. MATERIALS AND METHODS: A 70-year-old pancreatic head cancer patient was treated with C-ion RT on a clinical dose of 4.6 Gy (RBE), followed by AAPET, to verify beam ranges in C-ion RT. RESULTS: Positron emission tomography (PET) revealed low positron emitter concentrations at the distal side of the internal metals and in the aorta compared to the dose distribution of the treatment plan, indicating range shortening by internal metals in C-ion beams and positron emitter transportation by biofluids. CONCLUSION: AAPET may detect range shortening by internal metals in the trajectory and washout of intravascular positron emitter compared to plan dose distribution.

Fundamentals of Enzyme Kinetics: Michaelis-Menten and Non-Michaelis-Type (Atypical) Enzyme Kinetics.

This chapter will provide a general introduction to the kinetics of enzyme-catalyzed reactions, including a general discussion of catalysts, reaction rates, and binding constants. This section will be followed by a discussion of various types of enzyme kinetics observed in drug metabolism reactions. A large number of enzymatic reactions can be adequately described by Michaelis-Menten kinetics. The Michaelis-Menten equation represents a rectangular hyperbola, with a y-asymptote at the Vmax value. However, in other cases, more complex kinetic models are required to explain the observed data. Atypical kinetic profiles are believed to arise from the simultaneous binding of multiple molecules within the active site of the enzyme (Tracy and Hummel, Drug Metab Rev 36:231-242, 2004). Several cytochromes P450 (CYPs) have large active sites that enable binding of multiple molecules (Yano et al., J Biol Chem 279:38091-38094, 2004; Wester et al., J Biol Chem 279:35630-35637, 2004). Thus, atypical kinetics are not uncommon in in vitro drug metabolism studies.

The third model of Bax/Bak activation: a Bcl-2 family feud finally resolved?

Bax and Bak, two functionally similar, pro-apoptotic proteins of the Bcl-2 family, are known as the gateway to apoptosis because of their requisite roles as effectors of mitochondrial outer membrane permeabilization (MOMP), a major step during mitochondria-dependent apoptosis. The mechanism of how cells turn Bax/Bak from inert molecules into fully active and lethal effectors had long been the focal point of a major debate centered around two competing, but not mutually exclusive, models: direct activation and indirect activation. After intensive research efforts for over two decades, it is now widely accepted that to initiate apoptosis, some of the BH3-only proteins, a subclass of the Bcl-2 family, directly engage Bax/Bak to trigger their conformational transformation and activation. However, a series of recent discoveries, using previously unavailable CRISPR-engineered cell systems, challenge the basic premise that undergirds the consensus and provide evidence for a novel and surprisingly simple model of Bax/Bak activation: the membrane (lipids)-mediated spontaneous model. This review will discuss the evidence, rationale, significance, and implications of this new model.

Biochemical characterization of recombinant Penaeus vannamei trypsinogen.

Trypsinogens are the inactive precursors of trypsins (EC 3.4.21.4), which are digestive serine proteases. Despite knowing the properties of trypsins from Pacific white shrimp, Penaeus vannamei, the biochemical properties of shrimp trypsinogens including activation mechanisms and kinetics are unknown, due to difficulties isolating them from natural sources. In the present work, we describe the purification and biochemical characterization of four trypsinogen-like isoforms from recombinant P. vannamei trypsinogen, with a special emphasis on understanding its activation kinetics. The major trypsinogen-like isoform had an apparent molecular mass of 29 kDa. The other three forms of recombinant trypsinogen were: an N-glycosylated form of 32 kDa, a possibly O-glycosylated form of 41 kDa, and a likely double-chain form with a subunit of 23 kDa. The autoactivation profile of three-recombinant trypsinogen-like isoforms showed increased trypsin activity at a rate that was higher than that of bovine trypsinogen. This confirms the hypothesis proposed in the literature of a rapid trypsinogen autoactivation in the absence of aspartates in the activation peptide as it is for P. vannamei trypsinogen.

Novel Pathogenic PRSS1 Variant p.Glu190Lys in a Case of Chronic Pancreatitis.

Mutations in the PRSS1 (serine protease 1) gene encoding human cationic trypsinogen cause hereditary pancreatitis or may be associated with sporadic chronic pancreatitis. The mutations exert their pathogenic effect either by increasing intra-pancreatic trypsinogen activation (trypsin pathway) or by causing proenzyme misfolding and endoplasmic reticulum stress (misfolding pathway). Here we report a novel heterozygous c.568G>A (p.Glu190Lys) variant identified in a case with chronic pancreatitis. The parents of the index patient had no history of pancreatitis but were unavailable for genetic testing. Functional characterization revealed 2.5-fold increased autoactivation of the mutant trypsinogen relative to wild type. Unlike many other clinically relevant PRSS1 mutations, p.Glu190Lys did not alter the chymotrypsin C (CTRC)-dependent degradation of trypsinogen nor did it increase CTRC-mediated processing of the trypsinogen activation peptide. Cellular secretion of the mutant protein was unchanged indicating normal folding behavior. Based on the genetic and functional evidence, we classify the p.Glu190Lys PRSS1 variant as likely pathogenic, which stimulates autoactivation of cationic trypsinogen independently of CTRC.

Transcription factor ABI3 auto-activates its own expression during dehydration stress response.

The transcription factor abscisic acid insensitive 3 (ABI3) has recently been shown to mediate the dehydration stress response in nonseed and seed plants by regulation of several downstream genes. Here, we show how ABI3 autoregulates its transcription in response to dehydration stress signalling. Autoactivation is primarily through the Sph/RY element CATGCA present at the promoter region of ABI3. Along with other known cis-elements found at the ABI3 promoter, CATGCA remains occluded by nucleosomes during transcription repression. The nucleosomes tend to reposit during active transcription and are associated with several histone modifications, such as H3K9 and K27 acetylations and H3K4 trimethylation. This work thus, reveals the genetic and epigenetic essentials required for expression of the ABI3 gene, a crucial factor regulating dehydration stress signalling in Arabidopsis thaliana.