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Publikationen - Molekulare Signalverarbeitung

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Niemeyer, M.; Parra, J. O. F.; Calderón Villalobos, L. I. A.; An in vitro assay to recapitulate hormone-triggered and SCF-mediated protein ubiquitylation (Lois, L.M., Trujillo, M.). Methods Mol. Biol. 2581, 43-56, (2023) ISBN: 978-1-0716-2783-9 DOI: 10.1007/978-1-0716-2784-6_4

Signaling proteins trigger a sequence of molecular switches in the cell, which permit development, growth, and rapid adaptation to changing environmental conditions. SCF-type E3 ubiquitin ligases recognize signaling proteins prompting changes in their fate, one of these being ubiquitylation followed by degradation by the proteasome. SCFs together with their ubiquitylation targets (substrates) often serve as phytohormone receptors, responding and/or assembling in response to fluctuating intracellular hormone concentrations. Tracing and understanding phytohormone perception and SCF-mediated ubiquitylation of proteins could provide powerful clues on the molecular mechanisms utilized for plant adaptation. Here, we describe an adaptable in vitro system that uses recombinant proteins and enables the study of hormone-triggered SCF-substrate interaction and the dynamics of protein ubiquitylation. This system can serve to predict the requirements for protein recognition and to understand how phytohormone levels have the power to control protein fate.
Publikation

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A.; Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies Nat. Commun. 11, 2277, (2020) DOI: 10.1038/s41467-020-16147-2

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.
Preprints

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A.; Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies bioRxiv (2019) DOI: 10.1101/787770

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron′s vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.
Publikation

Naumann, C.; Müller, J.; Sakhonwasee, S.; Wieghaus, A.; Hause, G.; Heisters, M.; Bürstenbinder, K.; Abel, S.; The Local Phosphate Deficiency Response Activates Endoplasmic Reticulum Stress-Dependent Autophagy Plant Physiol. 179, 460-476, (2019) DOI: 10.1104/pp.18.01379

Inorganic phosphate (Pi) is often a limiting plant nutrient. In members of the Brassicaceae family, such as Arabidopsis (Arabidopsis thaliana), Pi deprivation reshapes root system architecture to favor topsoil foraging. It does so by inhibiting primary root extension and stimulating lateral root formation. Root growth inhibition from phosphate (Pi) deficiency is triggered by iron-stimulated, apoplastic reactive oxygen species generation and cell wall modifications, which impair cell-to-cell communication and meristem maintenance. These processes require LOW PHOSPHATE RESPONSE1 (LPR1), a cell wall-targeted ferroxidase, and PHOSPHATE DEFICIENCY RESPONSE2 (PDR2), the single endoplasmic reticulum (ER)-resident P5-type ATPase (AtP5A), which is thought to control LPR1 secretion or activity. Autophagy is a conserved process involving the vacuolar degradation of cellular components. While the function of autophagy is well established under nutrient starvation (C, N, or S), it remains to be explored under Pi deprivation. Because AtP5A/PDR2 likely functions in the ER stress response, we analyzed the effect of Pi limitation on autophagy. Our comparative study of mutants defective in the local Pi deficiency response, ER stress response, and autophagy demonstrated that ER stress-dependent autophagy is rapidly activated as part of the developmental root response to Pi limitation and requires the genetic PDR2-LPR1 module. We conclude that Pi-dependent activation of autophagy in the root apex is a consequence of local Pi sensing and the associated ER stress response, rather than a means for systemic recycling of the macronutrient.
Publikation

Hussain, H.; Ziegler, J.; Hause, G.; Wohlrab, J.; Neubert, R. H.; Quantitative Analysis of Free Amino Acids and Urea Derived from Isolated Corneocytes of Healthy Young, Healthy Aged, and Diseased Skin Skin Pharmacol. Physiol. 32, 94-100, (2019) DOI: 10.1159/000495992

Background/Aims: Free amino acids (FAAs) and urea, present inside the corneocytes, can be important indicators of skin condition. However, due to the lack of a standard extraction protocol for FAAs from corneocytes, conflicting research results have been reported. Therefore, the purpose of this study was (1) to standardize the extraction protocol and (2) to investigate FAA profiles in healthy young and healthy old volunteers, as well as in psoriasis and atopic dermatitis patients. Methods: Skin samples were collected from four groups (healthy young, healthy old, and psoriasis and atopic dermatitis patients) with 5 volunteers per group. Corneocytes were isolated and examined microscopically. FAAs and urea were extracted from the isolated corneocytes, and their amounts were quantified using LC-ESI/MS/MS (after derivatization with Fmoc-Cl) and colorimetric methods, respectively. Results: The micrographs of the corneocytes showed no morphological features attributable to age or disease conditions. The highest and lowest concentrations of total FAAs and urea were observed in the healthy old group and the healthy young group, respectively. Unlike the other FAAs and urea, citrulline was found at a higher level in the healthy young group than in the disease groups. Conclusion: This study suggests that the levels of FAAs and urea in the skin are affected by age and skin conditions (healthy/diseased). However, further studies are needed to show the effects of different skin conditions on the levels of FAAs and urea.
Publikation

Bagchi, R.; Melnyk, C. W.; Christ, G.; Winkler, M.; Kirchsteiner, K.; Salehin, M.; Mergner, J.; Niemeyer, M.; Schwechheimer, C.; Calderón Villalobos, L. I. A.; Estelle, M.; The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases EMBO J. 37, 255-268, (2018) DOI: 10.15252/embj.201797159

The cullin‐RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin‐conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
Publikation

Winkler, M.; Niemeyer, M.; Hellmuth, A.; Janitza, P.; Christ, G.; Samodelov, S. L.; Wilde, V.; Majovsky, P.; Trujillo, M.; Zurbriggen, M. D.; Hoehenwarter, W.; Quint, M.; Calderón Villalobos, L. I. A.; Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction Nat. Commun. 8, 15706, (2017) DOI: 10.1038/ncomms15706

Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.
Publikation

Bürstenbinder, K.; Möller, B.; Plötner, R.; Stamm, G.; Hause, G.; Mitra, D.; Abel, S.; The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus Plant Physiol. 173, 1692-1708, (2017) DOI: 10.1104/pp.16.01743

Calcium (Ca2+) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related calmodulin-like (CML) polypeptides are principal sensors of Ca2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67 DOMAIN (IQD) family emerged as possibly the largest class of CaM-interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis (Arabidopsis thaliana) differentially localize, using green fluorescent protein (GFP)-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane subdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca2+-CaM signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in Nicotiana benthamiana cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca2+ signaling at multiple cellular sites to regulate cell function, shape, and growth.
Publikation

Gasperini, D.; Acosta, I. F.; Farmer, E. E.; Cotyledon Wounding of Arabidopsis Seedlings Bio Protoc. 6, e1712, (2016) DOI: 10.21769/BioProtoc.1712

Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.
Publikation

Müller, J.; Toev, T.; Heisters, M.; Teller, J.; Moore, K.; Hause, G.; Dinesh, D.; Bürstenbinder, K.; Abel, S.; Iron-Dependent Callose Deposition Adjusts Root Meristem Maintenance to Phosphate Availability Dev. Cell 33, 216-230, (2015) DOI: 10.1016/j.devcel.2015.02.007

Plant root development is informed by numerous edaphic cues. Phosphate (Pi) availability impacts the root system architecture by adjusting meristem activity. However, the sensory mechanisms monitoring external Pi status are elusive. Two functionally interacting Arabidopsis genes, LPR1 (ferroxidase) and PDR2 (P5-type ATPase), are key players in root Pi sensing, which is modified by iron (Fe) availability. We show that the LPR1-PDR2 module facilitates, upon Pi limitation, cell-specific apoplastic Fe and callose deposition in the meristem and elongation zone of primary roots. Expression of cell-wall-targeted LPR1 determines the sites of Fe accumulation as well as callose production, which interferes with symplastic communication in the stem cell niche, as demonstrated by impaired SHORT-ROOT movement. Antagonistic interactions of Pi and Fe availability control primary root growth via meristem-specific callose formation, likely triggered by LPR1-dependent redox signaling. Our results link callose-regulated cell-to-cell signaling in root meristems to the perception of an abiotic cue.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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