C2 Domain Research Articles

Dancing Molecules: Group A bZIPs and PEBPs at the Heart of Plant Development and Stress Responses.

Group A basic leucine zipper (bZIP) transcription factors play critical roles in abscisic acid (ABA) signaling and plant development. In Arabidopsis thaliana, these factors are defined by a highly conserved core bZIP domain, and four conserved domains throughout their length: three at the N-terminus (C1 to C3) and a phosphorylatable C-terminal SAP motif located at the C4 domain. Initially, members such as ABI5 and ABFs were studied for their roles in ABA signaling during seed germination or stress responses. Later, a sub-clade of group A bZIPs, including FD, was found to play important roles in floral induction by interacting with the florigen FLOWERING LOCUS T (FT) at the shoot apical meristem. Recent research has expanded our understanding of these transcription factors by identifying intriguing parallels between those involved in ABA signaling and those promoting floral induction, and revealing dynamic interactions with FT and other phosphatidylethanolamine-binding proteins (PEBP) such as TERMINAL FLOWER 1. Studies in crop plants and non-model species demonstrate broader roles, functions, and molecular targets of group A bZIPs. This review highlights common features of group A bZIPs and their post-translational regulation in enabling the activation of gene regulatory networks with important functions in plant development and stress responses.

Validation of Machine Learning-assisted Screening of PKC Ligands: PKC Binding Affinity and Activation.

Protein kinase C (PKC) is a family of serine/threonine kinases, and PKC ligands have the potential to be therapeutic seeds for cancer, Alzheimer's disease, and human immunodeficiency virus infection. However, in addition to desired therapeutic effects, most PKC ligands also exhibit undesirable pro-inflammatory effects. The discovery of new scaffolds for PKC ligands is important for developing less inflammatory PKC ligands, such as bryostatins. We previously reported that machine learning combined with our knowledge of the pharmacophore yielded 15 PKC ligand candidates, but we did not evaluate their PKC binding affinities fully. In this paper, PKC binding affinities of four candidates were examined to assess their potential as PKC ligands and to validate machine learning-assisted screening. Although compound 3' did not bind to PKC C1 domains, 1a, 2', and 4a exhibited moderate PKC binding affinities, suggesting that machine learning-assisted screening is advantageous in identifying new PKC ligand scaffolds.

TC2N maintains stem cell-like characteristics to accelerate lung carcinogenesis by blockade of dual specificity protein phosphatase 3

BackgroundTandem C2 domains, nuclear (TC2N) is a protein that has been characterized to contain C2A domain, C2B domain, and a short C-terminus with a WHXL motif. In previous studies, we have uncovered the oncogenic role and mechanisms of TC2N in lung cancer: TC2N achieves this by inhibiting the p53 signaling pathway and activating the NF-kappaB signaling pathway. Beyond that, its precise function in tumorigenesis is not fully understood.MethodsTC2N-engineered mice model was used to assess the effect of TC2N knockout on normal lung and urethane-induced carcinogenesis. Tumor tissues of 395 lung cancer patients were subjected to tissue microarray and further assessed the associations of TC2N expression with tumor differentiation degree. The protein levels of TC2N and stem cell markers in cell lines and tissue specimens were monitored by WB and immunohistochemistry. In vitro cell assays were performed to assess the effect of TC2N ectopic expression on the stem cell-like characteristics of lung cancer cells. The downstream signaling pathway or target molecule of TC2N was mined using a combination of transcriptomics and proteomics, and the underlying mechanism was explored by WB and co-IP assays.ResultsHerein, TC2N appeared to have a strong effect in promoting lung tumorigenesis caused by urethane, whereas it seemed to lose its function in the normal lung. Meanwhile, we found that the functional differences of TC2N between lung tumor and normal lung were linked to its potential role in cancer cell stemness. Function-wise, TC2N overexpression maintained stem-like properties of lung cancer cell. Mechanism-wise, TC2N upregulated the phosphorylation of EGFR, ERK, STAT3 and FAK1 to activate these signaling pathways by the inhibition of DUSP3 phosphatase via a dual mechanism. Firstly, TC2N competes with EGFR, ERK, STAT3 and FAK1 for binding to DUSP3. This competition prevents these signaling molecules from being dephosphorylated by DUSP3, resulting in their sustained activation. Secondly, TC2N bind to DUSP3 and restrict the enzyme’s ability to dephosphorylate the signaling molecules.ConclusionsOverall, this study revealed a previously unknown role and mechanism of TC2N in the regulation of tumorigenesis and stemness in lung cancer cells.

Atomistic Mechanism of Lipid Membrane Binding for Blood Coagulation Factor VIII with Molecular Dynamics Simulations on a Microsecond Time Scale.

During the blood coagulation cascade, coagulation factor VIII (FVIII) is activated by thrombin to form activated factor VIII (FVIIIa). FVIIIa associates with platelet surfaces at the site of vascular damage to form an intrinsic tenase complex with activated factor IX. A working model for FVIII membrane binding involves the association of positively charged FVIII residues with negatively charged lipid headgroups and the burial of hydrophobic residues into the membrane interior. Currently, the atomic details of the FVIII lipid binding interactions and membrane orientation are lacking. This study reports residue-specific FVIII C domain interactions with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) in atomistic detail. Contact maps between residues in the C domains with different lipid moieties support prior structural data describing how the C domains associate with membranes through electrostatic and hydrophobic interactions. Solvent-accessible surface area analysis quantified the extent to which residues in the C1 and C2 domains bury into the membrane. Calculations of the potential energy between the C domains and DOPC and DOPS revealed an FVIII membrane-binding orientation that agrees with previous experimental data. This study expands our knowledge of the structural basis of FVIII membrane association, which may be critical for the development of next-generation FVIII replacement constructs with improved activity.

Structural Conformation and the Binding of Factor VIII R2159C (FVIII-Ise) Mutated in the C1 Domain to Phospholipid.

We previously identified a factor (F)VIII molecular defect associated with an R2159C mutation in the C1 domain (named "FVIII-Ise") together with undetectable FVIII antigen (FVIII:Ag) levels measured by two-site sandwich ELISA using an anti-C2 domain alloantibody (alloAb). The patient had clinically mild hemophilia A, and his reduced FVIII:C correlated with FVIII:Ag measured by ELISA using monoclonal antibodies (mAbs) with A2 and A2/B domain epitopes, suggesting that the R2159C mutation modified C2 domain antigenicity. To investigate functional and structural characteristics of the FVIII-R2159C mutant. ELISAs using a previous anti-C2 domain alloAb confirmed that the antigen level of recombinant FVIII-R2159C mutant prepared in BHK cells was 56% lower relative to wild-type (WT), consistent with our earlier reports. This anti-C2 domain alloAb competitively inhibited FVIII and anti-C1 domain mAb binding, indicating the involvement of specificity for C1 and C2 epitopes. The K m for FVIII-R2159C with FIXa or FX in the tenase complex was similar to that of FVIII-WT. Thrombin- and FXa-catalyzed cleavage reactions of FVIII-R2159C were similar to those of WT. The K d for FVIII-R2159C binding to phospholipids was moderately greater than for FVIII-WT, however, while there were no significant differences in von Willebrand factor binding. In silico molecular dynamic simulation analyses revealed subtle differences between FVIII-WT and FVIII-R2159C. The FVIII-R2159C mutation was not different from FVIII-WT in interactions with FIXa, FX, and thrombin, but reduced binding potential to phospholipids and to an anti-C1/C2 domain alloAb was evident apparently due to subtle changes in conformational structure.

Computational analysis of congenital heart disease associated SNPs: unveiling their impact on the gene regulatory system

Congenital heart disease (CHD) is a prevalent condition characterized by defective heart development, causing premature death and stillbirths among infants. Genome-wide association studies (GWASs) have provided insights into the role of genetic variants in CHD pathogenesis through the identification of a comprehensive set of single-nucleotide polymorphisms (SNPs). Notably, 90–95% of these variants reside in the noncoding genome, complicating the understanding of their underlying mechanisms. Here, we developed a systematic computational pipeline for the identification and analysis of CHD-associated SNPs spanning both coding and noncoding regions of the genome. Initially, we curated a thorough dataset of SNPs from GWAS-catalog and ClinVar database and filtered them based on CHD-related traits. Subsequently, these CHD-SNPs were annotated and categorized into noncoding and coding regions based on their location. To study the functional implications of noncoding CHD-SNPs, we cross-validated them with enhancer-specific histone modification marks from developing human heart across 9 Carnegie stages and identified potential cardiac enhancers. This approach led to the identification of 2,056 CHD-associated putative enhancers (CHD-enhancers), 38.9% of them overlapping with known enhancers catalogued in human enhancer disease database. We identified heart-related transcription factor binding sites within these CHD-enhancers, offering insights into the impact of SNPs on TF binding. Conservation analysis further revealed that many of these CHD-enhancers were highly conserved across vertebrates, suggesting their evolutionary significance. Utilizing heart-specific expression quantitative trait loci data, we further identified a subset of 63 CHD-SNPs with regulatory potential distributed across various cardiac tissues. Concurrently, coding CHD-SNPs were represented as a protein interaction network and its subsequent binding energy analysis focused on a pair of proteins within this network, pinpointed a deleterious coding CHD-SNP, rs770030288, located in C2 domain of MYBPC3 protein. Overall, our findings demonstrate that SNPs have the potential to disrupt gene regulatory systems, either by affecting enhancer sequences or modulating protein-protein interactions, which can lead to abnormal developmental processes contributing to CHD pathogenesis.

Evaluation of synaptotagmin-1 action models by all-atom molecular dynamics simulations.

Neurotransmitter release is triggered in microseconds by the two C2 domains of the Ca2+ sensor synaptotagmin-1 and by SNARE complexes, which form four-helix bundles that bridge the vesicle and plasma membranes. The synaptotagmin-1 C2B domain binds to the SNARE complex via a 'primary interface', but the mechanism that couples Ca2+-sensing to membrane fusion is unknown. Widespread models postulate that the synaptotagmin-1 Ca2+-binding loops accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but these models do not seem compatible with SNARE binding through the primary interface, which orients the Ca2+-binding loops away from the fusion site. To test these models, we performed molecular dynamics simulations of SNARE complexes bridging a vesicle and a flat bilayer, including the synaptotagmin-1 C2 domains in various configurations. Our data do not support the notion that insertion of the synaptotagmin-1 Ca2+-binding loops causes substantial membrane curvature or major perturbations of the lipid bilayers that could facilitate membrane fusion. We observed membrane bridging by the synaptotagmin-1 C2 domains, but such bridging or the presence of the C2 domains near the site of fusion hindered the action of the SNAREs in bringing the membranes together. These results argue against models predicting that synaptotagmin-1 triggers neurotransmitter release by inducing membrane curvature, perturbing bilayers or bridging membranes. Instead, our data support the hypothesis that binding via the primary interface keeps the synaptotagmin-1 C2 domains away from the site of fusion, orienting them such that they trigger release through a remote action.

A Genome-Wide Association Screen for Genes Affecting Leaf Trichome Development and Epidermal Metal Accumulation in Arabidopsis.

To identify novel genes engaged in plant epidermal development, we characterized the phenotypic variability of rosette leaf epidermis of 310 sequenced Arabidopsis thaliana accessions, focusing on trichome shape and distribution, compositional characteristics of the trichome cell wall, and histologically detectable metal ion distribution. Some of these traits correlated with cLimate parameters of our accession's locations of origin, suggesting environmental selection. A novel metal deposition pattern in stomatal guard cells was observed in some accessions. Subsequent GWAS analysis identified 1546 loci with protein sequence-altering SNPs associated with one or more traits, including 5 genes with previously reported relevant mutant phenotypes and 80 additional genes with known or predicted roles in relevant developmental and cellular processes. Some candidates, including GFS9/TT9, exhibited environmentally correlated allele distribution. Several large gene famiLies, namely DUF674, DUF784, DUF1262, DUF1985, DUF3741, cytochrome P450, receptor-Like kinases, Cys/His-rich C1 domain proteins and formins were overrepresented among the candidates for various traits, suggesting epidermal development-related functions. A possible participation of formins in guard cell metal deposition was supported by observations in available loss of function mutants. Screening of candidate gene lists against the STRING interactome database uncovered several predominantly nuclear protein interaction networks with possible novel roles in epidermal development.

Functional Characterization of the SHIP1-Domains Regarding Their Contribution to Inositol 5-Phosphatase Activity.

The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a multidomain protein consisting of two protein-protein interaction domains, the Src homology 2 (SH2) domain, and the proline-rich region (PRR), as well as three phosphoinositide-binding domains, the pleckstrin homology-like (PHL) domain, the 5-phosphatase (5PPase) domain, and the C2 domain. SHIP1 is commonly known for its involvement in the regulation of the PI3K/AKT signaling pathway by dephosphorylation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) at the D5 position of the inositol ring. However, the functional role of each domain of SHIP1 for the regulation of its enzymatic activity is not well understood. To determine the contribution of the individual domains to catalytic activity, the full-length protein was compared with truncated constructs lacking one or more domain(s), regarding the substrate turnover (kcat) and catalytic efficiency (kcat/Km) towards ci8-PtdIns(3,4,5)P3. With this approach, it was possible to verify the allosteric activation of SHIP1 mediated by the C2 domain as described previously, while the PHL domain seemed instead to have a negative effect regarding catalytic efficiency. The full-length SHIP1 clearly displayed the highest turnover and the second-highest catalytic efficiency, showing the role of the SH2 domain and PRR not only in protein-protein interactions but also in catalysis. The SH2 domain increased substrate turnover but negatively affected catalytic efficiency. The linker between the SH2 and the PHL domains decreased the turnover number but positively influenced the catalytic efficiency. The PRR increased both the substrate turnover and the protein's catalytic efficiency. The regression analysis of the Michaelis-Menten graph revealed SHIP1 to be an allosteric enzyme, with the PRR and the linker being the most involved domains in that regard. In summary, our data indicate a complex regulation of the enzymatic activity of SHIP1 by its individual domains. While the C2 domain and PRR at the carboxy-terminus have a positive effect on enzymatic activity, the SH2 and PHL domain at the amino-terminus inhibit catalytic efficiency.

Flow Cytometry Evaluation of Blood-Cell-Bound Surface FVIII in Hemophilia A and Thrombosis.

Hemophilia A (HA) is associated with FVIII coagulation insufficiency or inactivity leading to excessive bleeding. Elevated FVIII, on the contrary, is associated with thrombophilia, thrombosis, myocardial infarctions, and stroke. Active FVIII (aFVIII) uses its C2 domain to bind to blood cells' membranes, consequently carrying out its coagulative function. We developed a reliable flow cytometry (FC) method for FVIII detection that can be utilized for assessing surface-bound FVIII on leukocytes in different coagulation/clinical states; we analyzed 49 pediatric subjects, encompassing patients with HA, other coagulopathies, venous thrombosis, and normal coagulation. Interestingly, the total leukocyte surface FVIII showed a declining trend across thrombosis, normal, and hypo-coagulation states. As expected, the leukocytes of HA patients displayed significantly lower levels of cellular-surface FVIII in comparison to patients with thrombosis. However, no significant correlation was observed between circulating levels of FVIII in plasma and the levels of FVIII bound to leukocytes, indicating that the differences in FVIII surface binding are not directly proportional to the availability of FVIII in the circulation and suggesting a specific binding mechanism governing the interaction between FVIII and leukocytes. Intriguingly, when analyzing the distinct blood subpopulations, we observed that surface FVIII levels were significantly elevated in classical monocytes of thrombosis patients compared to HA patients, healthy controls, and patients with other coagulopathies. Our study highlights the reliability of our FC platform in assessing FVIII abundance on leukocytes' membranes across coagulation states. Monocytes, particularly in cases of thrombosis, exhibit active binding of FVIII on their surface, suggesting a potential role in the pathophysiology of thrombosis that requires further investigation.

Expression Analysis of Heavy-Chain-Only Antibodies in Cloudy Catshark and Japanese Bullhead Shark.

Heavy chain-only antibodies in sharks are called immunoglobulin new antigen receptors (IgNAR), consisting of one variable region (VNAR) and five constant regions (C1-C5). The variable region of IgNAR can be expressed as a monomer composed of a single domain, which has antigen specificity and is thus gaining attention as a next-generation antibody drug modality. In this study, we analyzed IgNAR of the cloudy catshark and Japanese bullhead shark, small demersal sharks available in the coastal waters of Japan. By analyzing the IgNAR gene sequence and comparing it with the constant regions of five other known shark species, high homology was observed in the C4 region. Consequently, we expressed the recombinant protein of the C4 domain from the cloudy catshark in E. coli, immunized rats, and produced antibodies. The obtained antiserum and mAbs recognized the C4 recombinant protein of the cloudy catshark, but reacted minimally with the plasma of non-immunized cloudy catsharks and instead reacted with the plasma of Japanese bullhead sharks. The results of this study imply that the protein expression levels of IgNAR in cloudy catsharks may be relatively lower compared to those in Japanese bullhead sharks, however, this interpretation remains to be determined through further studies.

Intracellular protein-lipid interactions drive presynaptic assembly prior to neurexin recruitment.

Neurexin cell-adhesion molecules regulate synapse development and function by recruiting synaptic components. Here, we uncover a mechanism for presynaptic assembly that precedes neurexin recruitment, mediated by interactions between cytosolic proteins and membrane phospholipids. Developmental imaging in C.elegans reveals that the intracellular active zone protein SYD-1 accumulates at nascent presynapses prior to its binding partner neurexin. Combining molecular dynamics simulations to model intrinsic interactions between SYD-1 and lipid bilayers with biochemical and invivo validation of these predictions, we find that PIP2-interacting residues in the SYD-1 C2 domain are required for active zone assembly. Genetic perturbation of a PIP2-generating enzyme disrupts synaptic SYD-1 accumulation, while the PIP2-interacting domain of mammalian RIM1 can compensate for the SYD-1 C2 domain, suggesting functional homology between these proteins. Finally, we propose that the evolutionarily conserved γ-neurexin isoform represents a minimal neurexin sequence that stabilizes nascent presynaptic assemblies, potentially a core function of this isoform.

Binding of therapeutic Fc-fused factor VIII to the neonatal Fc receptor at neutral pH associates with poor half-life extension.

Fusion of therapeutic proteins to the Fc fragment of human IgG1 promotes their FcRn-mediated recycling and subsequent extension in circulating half-life. However, different Fc-fused proteins, as well as antibodies with different variable domains but identical Fc, may differ in terms of extension in half-life. Here we compared the binding behaviour to FcRn of Fc-fused FVIII, Fc-fused FIX and two human monoclonal HIV-1 broadly-neutralizing IgG1, m66.6 and VRC01 with identical Fc. While all molecules bound FcRn at acidic pH, only rFVIIIFc and m66.6 interacted with FcRn at neutral pH. In silico modelling predicted a role for charged residues in the C1 and C2 domains of FVIII, and in the variable domains of m66.6, in the neutral binding to FcRn. Accordingly, mutations of key positively charged amino-acids in the FVIII C1C2 domains decreased the binding of the protein to FcRn at pH 7.4 in vitro and increased the half-life of rFVIIIFc in VWFKO mice. Our findings suggest that the removal of positively charged patches on Fc-fused proteins to ameliorate FcRn recycling without affecting therapeutic efficacy, may improve their pharmacokinetic properties.

FVIII peptides presented on HLA-DP and identification of an A3 domain peptide binding with high affinity to the commonly expressed HLA-DP4.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell dependent mechanism which includes antigen presenting cells (APCs), B- and T-helper lymphocytes. APCs present FVIII derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ Tcells. We previously established a mass spectrometry based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived dendritic cells (moDCs) from HLA-typed healthy donors. Using this method, we identified 176 to 1352 different HLA-DP presented peptides per donor, including 26 different FVIII derived peptides. The most frequently presented peptides derived from the A3, and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evaluated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLADP4. Interestingly, an A3 domain derived peptide bound with high affinity to HLA-DP4 positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.

Human genetic variation reveals FCRL3 is a lymphocyte receptor for Yersinia pestis.

Yersinia pestis is the gram-negative bacterium responsible for plague, one of the deadliest and most feared diseases in human history. This bacterium is known to infect phagocytic cells, such as dendritic cells and macrophages, but interactions with non-phagocytic cells of the adaptive immune system are frequently overlooked despite the importance they likely hold for human infection. To discover human genetic determinants of Y. pestis infection, we utilized nearly a thousand genetically diverse lymphoblastoid cell lines in a cellular genome-wide association study method called Hi-HOST (High-throughput Human in-vitrO Susceptibility Testing). We identified a nonsynonymous SNP, rs2282284, in Fc receptor like 3 (FCRL3) associated with bacterial invasion of host cells (p=9×10-8). FCRL3 belongs to the immunoglobulin superfamily and is primarily expressed in lymphocytes. rs2282284 is within a tyrosine-based signaling motif, causing an asparagine-to-serine mutation (N721S) in the most common FCRL3 isoform. Overexpression of FCRL3 facilitated attachment and invasion of non-opsonized Y. pestis. Additionally, FCRL3 colocalized with Y. pestis at sites of cellular attachment, suggesting FCRL3 is a receptor for Y. pestis. These properties were variably conserved across the FCRL family, revealing molecular requirements of attachment and invasion, including an Ig-like C2 domain and a SYK interaction motif. Direct binding was confirmed with purified FCRL5 extracellular domain. Following attachment, invasion of Y. pestis was dependent on SYK and decreased with the N721S mutation. Unexpectedly, this same variant is associated with risk of chronic hepatitis C virus infection in BioBank Japan. Thus, Y. pestis hijacks FCRL proteins, possibly taking advantage of an immune receptor to create a lymphocyte niche during infection.

Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1.

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense-core vesicles (DCVs) and synaptic vesicles to the plasma membrane and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1's roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.

MCTP controls nucleocytoplasmic partitioning of AUXIN RESPONSE FACTORs during lateral root development.

The plant hormone auxin orchestrates almost all aspects of plant growth and development. AUXIN RESPONSE FACTORs (ARFs) control the transcription of auxin-responsive genes, forming cytoplasmic condensates to modulate auxin sensitivity and diversify auxin response regulation. However, the dynamic control of ARF distribution across different subcellular compartments remains largely obscure. Here, we show that three MULTIPLE C2 DOMAIN AND TRANSMEMBRANE REGION PROTEINs (MCTPs), MCTP3, MCTP4, and MCTP6, control ARF nucleocytoplasmic partitioning and determine lateral root development. MCTP3/4/6 are highly expressed in lateral roots and specifically interact with ARF7 and ARF19 to dissolve their cytoplasmic condensates. This promotes ARF nuclear localization in lateral root primordia and enhances auxin signaling during lateral root formation. Our findings confer MCTP as a key switch to modulate auxin responses and outline an MCTP-ARF signaling cascade that is crucial for the establishment of the plant root system.

The small ARF-like 2 GTPase TITAN5 is linked with the dynamic regulation of IRON-REGULATED TRANSPORTER 1.

Iron acquisition is crucial for plants. The abundance of IRON-REGULATED TRANSPORTER 1 (IRT1) is controlled through endomembrane trafficking, a process that requires small ARF-like GTPases. Only few components that are involved in the vesicular trafficking of specific cargo are known. Here, we report that the ARF-like GTPase TITAN5 (TTN5) interacts with the large cytoplasmic variable region and protein-regulatory platform of IRT1. Heterozygous ttn5-1 plants can display reduced root iron reductase activity. This activity is needed for iron uptake via IRT1. Fluorescent fusion proteins of TTN5 and IRT1 colocalize at locations where IRT1 sorting and cycling between the plasma membrane and the vacuole are coordinated. TTN5 can also interact with peripheral membrane proteins that are components of the IRT1 regulation machinery, like the trafficking factor SNX1, the C2 domain protein EHB1 and the SEC14-GOLD protein PATL2. Hence, the link between iron acquisition and vesicular trafficking involving a small GTPase of the ARF family opens up the possibility to study the involvement of TTN5 in nutritional cell biology and the endomembrane system.

The Role of Protein-Lipid Interactions in Priming the Bacterial Translocon.

Protein-lipid interactions demonstrate important regulatory roles in the function of membrane proteins. Nevertheless, due to the semi-liquid nature and heterogeneity of biological membranes, and dissecting the details of such interactions at high resolutions continues to pose a major challenge to experimental biophysical techniques. Computational techniques such as molecular dynamics (MD) offer an alternative approach with both temporally and spatially high resolutions. Here, we present an extensive series of MD simulations focused on the inner membrane protein YidC (PDB: 6AL2) from Escherichia coli, a key insertase responsible for the integration and folding of membrane proteins. Notably, we observed rare lipid fenestration events, where lipids fully penetrate the vestibule of YidC, providing new insights into the lipid-mediated regulation of protein insertion mechanisms. Our findings highlight the direct involvement of lipids in modulating the greasy slide of YidC and suggest that lipids enhance the local flexibility of the C1 domain, which is crucial for recruiting substrate peptides. These results contribute to a deeper understanding of how protein-lipid interactions facilitate the functional dynamics of membrane protein insertases, with implications for broader studies of membrane protein biology.

The effects of WWP1 overexpression on the golgi apparatus stress response and proteoglycan production in adipocytes

White adipocytes are a major component of white adipose tissue (WAT) and help to maintain systemic metabolic homeostasis by storing energy and secreting adipokines. In mice deficient in the protein WWP1 (WW domain-containing E3 ubiquitin protein ligase 1), oxidative stress in adipocytes increases but insulin resistance induced by obesity improves. However, the specific roles of WWP1 in adipocytes remain unclear. Here, we show that in 3T3L1 adipocytes, WWP1 localized in the Golgi apparatus via its C2 domain, where it protected the Golgi apparatus from monensin-induced disruption. By contrast, WWP1 knockdown by short hairpin RNA failed to protect the Golgi apparatus and enhanced Golgi apparatus disruption by monensin. The Golgi apparatus acts as a central organelle to establish accurate protein glycosylation of proteoglycans containing glycosaminoglycans, including chondroitin sulfate and heparan sulfate (HS). WWP1 overexpression increased chondroitin sulfate and HS levels, whereas WWP1 knockdown decreased them. Furthermore, obesity-related increases in HS were prevented by WWP1 knockout in adipose tissue. In summary, our results demonstrate a novel role for WWP1 in maintaining Golgi apparatus morphology and proteoglycan synthesis in adipocytes.