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CULLIN4 (CUL4) RING ligase (CRL4) complexes contain a CUL4 scaffold protein, associated to RBX1 and to DDB1 proteins and have traditionally been associated to protein degradation events. Through DDB1, these complexes can associate with numerous DCAF proteins, which directly interact with specific targets promoting their ubiquitination and subsequent degradation by the proteasome. A characteristic feature of the majority of DCAF proteins that associate with DDB1 is the presence of the DWD motif. DWD-containing proteins sum up to 85 in the plant model species Arabidopsis. In the last decade, numerous Arabidopsis DWD proteins have been studied and their molecular functions uncovered. Independently of whether their association with CRL4 has been confirmed or not, DWD proteins are often found as components of additional multimeric protein complexes that play key roles in essential nuclear events. For most of them, the significance of their complex partnership is still unexplored. Here, we summarize recent findings involving both confirmed and putative CRL4-associated DCAF proteins in regulating nuclei architecture remodelling, DNA damage repair, histone post-translational modification, mRNA processing and export, and ribosome biogenesis, that definitely have an impact in gene expression and de novo protein synthesis. We hypothesized that, by maintaining accurate levels of regulatory proteins through targeted degradation and transcriptional control, CRL4 complexes help to surveil nuclear processes essential for plant development and survival.

Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis. However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47. Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance.

To cope with growth in low-phosphate (Pi) soils, plants have evolved adaptive responses that involve both developmental and metabolic changes. PHOSPHATE STARVATION RESPONSE 1 (PHR1) and related transcription factors play a central role in the control of Pi starvation responses (PSRs). How Pi levels control PHR1 activity, and thus PSRs, remains to be elucidated. Here, we identify a direct Pi-dependent inhibitor of PHR1 in Arabidopsis , SPX1, a nuclear protein that shares the SPX domain with yeast Pi sensors and with several Pi starvation signaling proteins from plants. Double mutation of SPX1 and of a related gene, SPX2, resulted in molecular and physiological changes indicative of increased PHR1 activity in plants grown in Pi-sufficient conditions or after Pi refeeding of Pi-starved plants but had only a limited effect on PHR1 activity in Pi-starved plants. These data indicate that SPX1 and SPX2 have a cellular Pi-dependent inhibitory effect on PHR1. Coimmunoprecipitation assays showed that the SPX1/PHR1 interaction in planta is highly Pi-dependent. DNA-binding and pull-down assays with bacterially expressed, affinity-purified tagged SPX1 and ΔPHR1 proteins showed that SPX1 is a competitive inhibitor of PHR1 binding to its recognition sequence, and that its efficiency is highly dependent on the presence of Pi or phosphite, a nonmetabolizable Pi analog that can repress PSRs. The relative strength of the SPX1/PHR1 interaction is thus directly influenced by Pi, providing a link between Pi perception and signaling. Significance When P levels are low, plants activate an array of adaptive responses to increase efficient acquisition and use of phosphate (Pi), the form in which P is preferentially absorbed, and to protect themselves from Pi starvation stress. Considerable progress has been made recently in dissecting the plant Pi starvation signaling pathway. Nonetheless, little is known as to how Pi levels are perceived by plants. Here, we identify the nuclear protein SPX1 as a Pi-dependent inhibitor of DNA binding by PHOSPHATE STARVATION RESPONSE 1 (PHR1), a master regulator of Pi starvation responses. We show that the Pi dependence of SPX1 inhibition of PHR1 activity can be recreated in vitro using purified proteins, which indicates that the SPX1/PHR1 module links Pi sensing and signaling.

Plants respond to different stresses by inducing or repressing transcription of partially overlapping sets of genes. In Arabidopsis, the PHR1 transcription factor (TF) has an important role in the control of phosphate (Pi) starvation stress responses. Using transcriptomic analysis of Pi starvation in phr1, and phr1 phr1-like (phl1) mutants and in wild type plants, we show that PHR1 in conjunction with PHL1 controls most transcriptional activation and repression responses to phosphate starvation, regardless of the Pi starvation specificity of these responses. Induced genes are enriched in PHR1 binding sequences (P1BS) in their promoters, whereas repressed genes do not show such enrichment, suggesting that PHR1(-like) control of transcriptional repression responses is indirect. In agreement with this, transcriptomic analysis of a transgenic plant expressing PHR1 fused to the hormone ligand domain of the glucocorticoid receptor showed that PHR1 direct targets (i.e., displaying altered expression after GR:PHR1 activation by dexamethasone in the presence of cycloheximide) corresponded largely to Pi starvation-induced genes that are highly enriched in P1BS. A minimal promoter containing a multimerised P1BS recapitulates Pi starvation-specific responsiveness. Likewise, mutation of P1BS in the promoter of two Pi starvation-responsive genes impaired their responsiveness to Pi starvation, but not to other stress types. Phylogenetic footprinting confirmed the importance of P1BS and PHR1 in Pi starvation responsiveness and indicated that P1BS acts in concert with other cis motifs. All together, our data show that PHR1 and PHL1 are partially redundant TF acting as central integrators of Pi starvation responses, both specific and generic. In addition, they indicate that transcriptional repression responses are an integral part of adaptive responses to stress.

Urea cycle disorders (UCDs) are inborn errors of ammonia detoxification/arginine synthesis due to defects affecting the catalysts of the Krebs-Henseleit cycle (five core enzymes, one activating enzyme and one mitochondrial ornithine/citrulline antiporter) with an estimated incidence of 1:8.000. Patients present with hyperammonemia either shortly after birth (~50%) or, later at any age, leading to death or to severe neurological handicap in many survivors. Despite the existence of effective therapy with alternative pathway therapy and liver transplantation, outcomes remain poor. This may be related to underrecognition and delayed diagnosis due to the nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity. These guidelines aim at providing a trans-European consensus to: guide practitioners, set standards of care and help awareness campaigns. To achieve these goals, the guidelines were developed using a Delphi methodology, by having professionals on UCDs across seven European countries to gather all the existing evidence, score it according to the SIGN evidence level system and draw a series of statements supported by an associated level of evidence. The guidelines were revised by external specialist consultants, unrelated authorities in the field of UCDs and practicing pediatricians in training. Although the evidence degree did hardly ever exceed level C (evidence from non-analytical studies like case reports and series), it was sufficient to guide practice on both acute and chronic presentations, address diagnosis, management, monitoring, outcomes, and psychosocial and ethical issues. Also, it identified knowledge voids that must be filled by future research. We believe these guidelines will help to: harmonise practice, set common standards and spread good practices with a positive impact on the outcomes of UCD patients.

CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDBI-ASSOCIATEDI (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABAmediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.

We present novel fluorescent cholesteryl probes (CNDs) with a modular design based on the solvatochromic 1,8-phthalimide scaffold. We have explored how different modules—linkers and head groups—affect the ability of these probes to integrate into lipid membranes and how they distribute intracellularly in mouse astrocytes and fibroblasts targeting lysosomes and lipid droplets. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes. Molecular dynamics simulations and lipid partitioning using giant unilamellar vesicles showed how these analogs behave in model membranes compared to cholesterol. Live-cell imaging demonstrated distinct staining patterns and cellular uptake behaviors, further validating the utility of these probes in biological systems. We compared the empirical results with those of BODIPY-cholesterol, a well-regarded fluorescent cholesterol analog. The internalization efficiency of fluorescent CND probes varies in different cell types and is affected mainly by the head groups. Our results demonstrate that the modular design significantly simplifies the creation of fluorescent cholesteryl probes bearing distinct spectral, biophysical, and cellular targeting features. It is a valuable toolkit for imaging in live cells, measuring cellular membrane dynamics, and studying cholesterol-related processes.

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.

Significant shortcomings in the global response to COVID-19 have revealed a longstanding reality: the current international health and intellectual property (IP) laws and practices fail to deliver equitable access to medical countermeasures (ie, vaccines, therapeutics, diagnostics and personal protective equipment) for global health crises. Since 2020, governments worldwide have spent US$5.6 billion on COVID-19 research and development (R&D) and US$45 billion on advanced purchase agreements.1 Yet, these funding agreements have not enabled the transfer of manufacturing know-how to scale up vaccine production and make access more equal. [...]large parts of the world were left unprotected from the virus, allowing the rise of new variants and prolonging the pandemic for everyone. Two, while the World Trade Organization (WTO) Trade-Related Aspects of Intellectual Property Rights Agreement has policy space to protect public health and to promote access to medicines for all, compliance with the legal texts requires a sophisticated understanding of both IP and trade law, and an ability to resist pressure from trading partners and rights holders.4 Compulsory licensing of patents is a powerful legal tool to deal with patent barriers to access to health technologies, in the absence of voluntary licenses, but have scarcely been used during the COVID-19 pandemic. A critical component is to obtain sufficient rights to ensure that patents, data, know-how and biological resources can be shared as needed to replicate the innovation by qualified entities, subject to appropriate safeguards and conditions, including when appropriate, remuneration.

The regulation of protein abundance of receptors and transporters at the plasma membrane is important for proper signaling in many biological pathways. The removal of plasma membrane proteins can occur via the endocytic protein degradation pathway, in which posttranslational modification by ubiquitin plays a key role. The activity of ubiquitinating and deubiquitinating enzymes can determine the ubiquitination status of a given target protein, and it has been shown that both classes of enzymes have important physiological roles. However, how these enzymes themselves are regulated at the molecular level has not yet been completely understood. In this study, we report a possible mechanism by which the deubiquitinating enzyme AMSH3 is regulated by its interacting protein, apoptosis-linked gene-2 interacting protein X (ALIX), in Arabidopsis . Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.