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Phase separation of biomolecules can facilitate their spatiotemporally regulated self-assembly within living cells. Due to the selective yet dynamic exchange of biomolecules across condensate interfaces, condensates can function as reactive hubs by concentrating enzymatic components for faster kinetics. The principles governing this dynamic exchange between condensate phases, however, are poorly understood. In this work, we systematically investigate the influence of client–sticker interactions on the exchange dynamics of protein molecules across condensate interfaces. We show that increasing affinity between a model protein scaffold and its client molecules causes the exchange of protein chains between the dilute and dense phases to slow down and that beyond a threshold interaction strength, this slowdown in exchange becomes substantial. Investigating the impact of interaction symmetry, we found that chain exchange dynamics are also considerably slower when client molecules interact equally with different sticky residues in the protein. The slowdown of exchange is due to a sequestration effect, by which there are fewer unbound stickers available at the interface to which dilute phase chains may attach. These findings highlight the fundamental connection between client–scaffold interaction networks and condensate exchange dynamics. [ABSTRACT FROM AUTHOR]

• Expression of GABAergic markers and scaffolding proteins increases in the early stages of development. • Oxytocin increases GABAergic synaptic puncta in the piriform cortex. • Oxytocin did not affect neurite branching in GABAergic neurons isolated from the olfactory bulb. Oxytocin affects social recognition, interactions, and behavior in adults. Despite growing data on the role of oxytocin in the sensory systems, its effects on early olfactory system development remain poorly understood. The present study aimed to investigate the developmental impact of oxytocin on selected parameters of the GABAergic system in olfactory brain regions. We found a significant increase in the expression of GABAergic markers and scaffolding proteins in the olfactory bulb during the early stages of development in both male and female rats, regardless of oxytocin treatment administered on postnatal days 2 and 3 (P2 and P3, 5 µg/pup). Oxytocin administration markedly reduced the expression of the scaffolding protein Gephyrin in male rats and it led to a significant increase in the number of GABAergic synaptic puncta in the piriform cortex of male rats at P5, P7, and P9. Our data suggest that the developmental action of oxytocin in relation to the GABAergic system may represent a mechanism by which the plasticity and maturation of olfactory brain regions are regulated. [ABSTRACT FROM AUTHOR]

Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites. Synopsis: Cytokinetic abscission in Toxoplasma gondii is dependent on the accumulation of membrane material at the division plane. Instead of the ESCRT-III machinery, this parasite and other apicomplexans have evolved a specific set of Daughter Cell Scaffold proteins that include PP2A-2 subunits and are specifically required for this process. The dynamic localization of DCS and PP2A-2 suggests that they act early during division, while affecting the daughter cell separation at the end of the endodyogeny. Expression of all DCS proteins and PP2A-2 subunits is required for proper cellular abscission. Depletion of DCS or PP2A-2 proteins leads to the formation of a parasite syncytium that is able to egress but shows defects in motility and invasion. Syncytia consisting of two conjoined parasites can invade the host cell and establish a new lytic cycle. A set of five Toxoplasma gondii proteins is crucial for the buildup of the plasma membrane at the division plane, ensuring the successful completion of cellular abscission. [ABSTRACT FROM AUTHOR]

FKBP51, also known as FK506‐binding protein 51, is a molecular chaperone and scaffolding protein with significant roles in regulating hormone signaling and responding to stress. Genetic variants in FKBP5, which encodes FKBP51, have been implicated in a growing number of neuropsychiatric disorders, which has spurred efforts to target FKBP51 therapeutically. However, the molecular mechanisms and sub‐anatomical regions influenced by FKBP51 in these disorders are not fully understood. In this study, we aimed to examine the impact of Fkbp5 ablation using circadian phenotyping and molecular analyses. Our findings revealed that the lack of FKBP51 did not significantly alter circadian rhythms, as detected by wheel‐running activity, but did offer protection against stress‐mediated disruptions in rhythmicity in a sex‐dependent manner. Protein changes in Fkbp5 KO mice, as measured by histology and proteomics, revealed alterations in a brain region‐ and sex‐dependent manner. Notably, regardless of sex, aged Fkbp5 KOs showed elevated MYCBP2, FBXO45, and SPRYD3 levels, which are associated with neuronal‐cell adhesion and synaptic integrity. Additionally, pathways such as serotonin receptor signaling and S100 family signaling were differentially regulated in Fkbp5 KO mice. Weighted protein correlation network analysis identified protein networks linked with synaptic transmission and neuroinflammation. The information generated by this work can be used to better understand the molecular changes in the brain during aging and in the absence of Fkbp5, which has implications for the continued development of FKBP51‐focused therapeutics for stress‐related disorders. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase 3β (GSK-3β) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component β-catenin for phosphorylation by GSK-3β and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3β bound to a phosphoprimed β-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of β-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3β with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3β active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3β and facilitate β-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution. Editor's summary: The kinase GSK-3β is a potential therapeutic target in various neurodegenerative diseases. After phosphorylation of the Wnt signaling component β-catenin by an upstream kinase (phosphopriming), β-catenin is subsequently phosphorylated by GSK-3β and targeted for degradation. Enos et al. determined structures of GSK-3β bound to phosphoprimed or phosphomimetic β-catenin and to the scaffold Axin, which showed that substrate positioning from these interactions was not sufficient for optimal catalysis. However, phosphorylation of phosphoprimed β-catenin was more rapid than that of the phosphomimetic peptide, highlighting the importance of native substrate phosphopriming for catalytic function. —John F. Foley [ABSTRACT FROM AUTHOR]

In cells, microtubules (MTs) assemble from α/β‐tubulin subunits at nucleation sites containing the γ‐tubulin ring complex (γ‐TuRC). Within the γ‐TuRC, exposed γ‐tubulin molecules act as templates for MT assembly by interacting with α/β‐tubulin. The vertebrate γ‐TuRC is scaffolded by γ‐tubulin‐interacting proteins GCP2‐6 arranged in a specific order. Interestingly, the γ‐tubulin molecules in the γ‐TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ‐TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ‐TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α‐tubulin mutant, resulted in a closed, cylindrical γ‐TuRC showing canonical interactions between all γ‐tubulin molecules and α/β‐tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ‐TuRC, with some γ‐tubulin molecules failing to align with α/β‐tubulin. This review discusses these outcomes, along with the broader implications. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are attractive therapeutic agents in a wide range of human disorders that bind specifically to their target through their complementary‐determining regions (CDRs). Small proteins with structurally preserved CDRs are promising antibodies mimetics. In this in silico study, we presented new antibody mimetics against the cancer marker epidermal growth factor receptor (EGFR) created by the CDRs grafting technique. Ten potential graft acceptor sites that efficiently immobilize the grafted CDR loops were selected from three small protein scaffolds using a computer. The three most involved CDR loops in antibody‐receptor interactions extracted from panitumumab antibody against the EGFR domain III crystal structure were then grafted to the selected scaffolds through the loop randomization technique. The combination of three CDR loops and 10 grafting sites revealed that three of the 36 combinations showed specific binding to EGFR DIII by binding energy calculations. Thus, the present strategy and selected small protein scaffolds are promising tools in the design of new binders against EGFR with high binding energy. [ABSTRACT FROM AUTHOR]

β‐arrestin2, a member of the arrestin family, mediates the desensitization and internalization of most G protein‐coupled receptors (GPCRs) and functions as a scaffold protein in signalling pathways. Previous studies have demonstrated that β‐arrestin2 expression is dysregulated in malignant tumours, fibrotic diseases, cardiovascular diseases and metabolic diseases, suggesting its pathological roles. Transcription and post‐transcriptional modifications can affect the expression of β‐arrestin2. Furthermore, post‐translational modifications, such as phosphorylation, ubiquitination, SUMOylation and S‐nitrosylation affect the cellular localization of β‐arrestin2 and its interaction with downstream signalling molecules, which further regulate the activity of β‐arrestin2. This review summarizes the structure and function of β‐arrestin2 and reveals the mechanisms involved in the regulation of β‐arrestin2 at multiple levels. Additionally, recent studies on the role of β‐arrestin2 in some major diseases and its therapeutic prospects have been discussed to provide a reference for the development of drugs targeting β‐arrestin2. [ABSTRACT FROM AUTHOR]

Proteins are one of the most prevalent components of the extracellular matrix, which are used in bone tissue engineering for construction of scaffolds. Due to their biocompatibility, biodegradability, non-immunogenicity and having cell motifs (cell recognition sites), they are capable of providing cell adhesion and favorable scaffold-cell interactions. However, proteins have crucial disadvantages, including lack of mechanical strength required for bone regeneration, as well as rapid degradation rate, which have restricted their implementation as a sole component in the construction of scaffolds. To overcome such disadvantages and at the same time, benefit from their properties, proteins are combined with other materials, such as carbohydrates and synthetic polymers and glass-ceramides. This review summarizes different types of protein hybrid scaffolds for bone tissue engineering and also discusses the function and significance of proteins in bone regeneration. [ABSTRACT FROM AUTHOR]

Serine/threonine kinase receptor-associated protein (STRAP) serves as a scaffold protein and is engaged in a variety of cellular activities, although its importance in antiviral innate immunity is unknown. We discovered that STRAP works as an interferon (IFN)-inducible positive regulator, facilitating type I IFN signaling during pseudorabies virus infection. Mechanistically, STRAP interacts with TBK1 to activate type I IFN signaling. Both the CT and WD40 7 − 6 domains contribute to the function of STRAP. Furthermore, TBK1 competes with PRV-UL50 for binding to STRAP, and STRAP impedes the degradation of TBK1 mediated by PRV-UL50, thereby increasing the interaction between STRAP and TBK1. Overall, these findings reveal a previously unrecognized role for STRAP in innate antiviral immune responses during PRV infection. STRAP could be a potential therapeutic target for viral infectious diseases. [ABSTRACT FROM AUTHOR]

The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines. [ABSTRACT FROM AUTHOR]

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders. Neutrophils contribute to the pathogenesis of chronic inflammatory diseases, but the mechanisms underlying their recruitment to inflamed skin are unclear. This study shows that repeated acute stress to the skin amplifies a neutrophil infiltration response involving keratin 17 and PKCα. [ABSTRACT FROM AUTHOR]

Background Objective Methods Results Conclusions There are more than 30 immunoglobulin G (IgG) autoantibody biomarkers of autoimmune cerebellar ataxia reported.To describe a novel IgG biomarker targeting neurobeachin, a scaffolding protein integral to synapse formation and function.Clinical and laboratory‐based assessment was used for a novel diagnostic biomarker of autoimmune ataxia.A 46‐year‐old woman had ataxia onset followed by bilateral knee pain, attributable to rheumatoid arthritis. She was negative for standard neural antibodies. Her ataxia stabilized and tremor initially improved after corticosteroid treatment, but she progressed to wheelchair‐dependence (on methotrexate and then rituximab) over 4 years, in keeping with the trajectory encountered by many with autoimmune ataxia. In contrast, her inflammatory arthritis remitted with immunotherapy. Neurobeachin‐IgG was unmasked in serum and cerebrospinal fluid using protein arrays and confirmed using neurobeachin‐specific confocal immunofluorescence and Western blot.Neurobeachin‐IgG is a new neural antibody biomarker of cerebellar ataxia among a growing antibody repertoire, collectively common but often individually rare. © 2024 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

The development of novel therapeutic proteins is a lengthy and costly process, with an average attrition rate of 91% (Thomas et al. Clinical Development Success Rates and Contributing Factors 2011–2020, 2021). To increase the probability of success and ensure robust drug supply beyond approval, it is essential to assess the developability profile of new potential drug candidates as early and broadly as possible in development (Jain et al. MAbs, 2023. https://doi.org/10.1016/j.copbio.2011.06.002). Predicting these properties in silico is expected to be the next leap in innovation as it would enable significantly reduced development timelines combined with broader screens at lower costs. However, developing predictive algorithms typically requires substantial datasets generated under very defined conditions, a limiting factor especially for new classes of therapeutic proteins that hold immense clinical promise. Here we describe a strategy for assessing the developability of a novel class of small therapeutic Anticalin® proteins using machine learning in conjunction with a knowledge-driven approach. The knowledge-driven approach considers developability attributes such as aggregation propensity, charge variants, immunogenicity, specificity, thermal stability, hydrophobicity, and potential post-translational modifications, to calculate a holistic developability score. Based on sequence-derived descriptors as input parameters we established novel statistical models designed to predict the developability scores for Anticalin proteins. The best models yielded low root mean square errors across the entire dataset and were further validated by removing input data from individual screening campaigns and predicting developability scores for those drug candidates. The adoption of the described workflow will enable significantly streamlined preclinical development of Anticalin drug candidates and could potentially be applied to other therapeutic protein scaffolds. [ABSTRACT FROM AUTHOR]

Molecular catalysts based on abundant elements that function in neutral water represent an essential component of sustainable hydrogen production. Artificial hydrogenases based on protein-inorganic hybrids have emerged as an intriguing class of catalysts for this purpose. We have prepared a novel artificial hydrogenase based on cobaloxime bound to a de novo three alpha-helical protein, α3C, via a pyridyl-based unnatural amino acid. The functionalized de novo protein was characterised by UV-visible, CD, and EPR spectroscopy, as well as MALDI spectrometry, which confirmed the presence and ligation of cobaloxime to the protein. The new de novo enzyme produced hydrogen under electrochemical, photochemical and reductive chemical conditions in neutral water solution. A change in hydrogen evolution capability of the de novo enzyme compared with native cobaloxime was observed, with turnover numbers around 80% of that of cobaloxime, and hydrogen evolution rates of 40% of that of cobaloxime. We discuss these findings in the context of existing literature, how our study contributes important information about the functionality of cobaloximes as hydrogen evolving catalysts in protein environments, and the feasibility of using de novo proteins for development into artificial metalloenzymes. Small de novo proteins as enzyme scaffolds have the potential to function as upscalable bioinspired catalysts thanks to their efficient atom economy, and the findings presented here show that these types of novel enzymes are a possible product. [ABSTRACT FROM AUTHOR]

Summary Light and brassinosteroids (BR) are indispensable for plant growth and control cell division in the apical meristem. However, how external light signals cooperate with internal brassinosteroids to program root meristem development remains elusive. We reveal that the photoreceptor phytochrome B (phyB) guides the scaffold protein RACK1 to coordinate BR signaling for maintaining root meristematic activity. phyB and RACK1 promote early root meristem development. Mechanistically, RACK1 could reinforce the phyB–SPA1 association by interacting with both phyB and SPA1, which indirectly affects COP1‐dependent RACK1 degradation, resulting in the accumulation of RACK1 in roots. Subsequently, RACK1 interacts with BES1 to repress its DNA‐binding activity toward the target gene CYCD3;1, leading to the release of BES1‐mediated inhibition of CYCD3;1 transcription, and hence the promotion of root meristem development. Our study provides mechanistic insights into the regulation of root meristem development by combination of light and phytohormones signals through the photoreceptors and scaffold proteins. [ABSTRACT FROM AUTHOR]

Limited effective targets have challenged the treatment of oral squamous cell carcinoma (OSCC). Casein kinase 2 interacting protein 1 (CKIP‐1) is a scaffold protein involved in various diseases. However, the role of CKIP‐1 in OSCC remains unclear. The aim of this study was to explore the regulatory role of CKIP‐1 in OSCC, as well as the involved mechanism. First, higher expression of CKIP‐1 in OSCC tissues and cell lines were found. Series of gain‐ and loss‐of‐function experiments demonstrated suppressed malignant behaviours and enhanced apoptosis of OSCC cells when CKIP‐1 was silenced. Also, inhibited tumour growth in CKIP‐1‐silenced group were proved. Further, mitochondrial transcription factor A (TFAM) downregulation, increased ROS production, decreased mitochondrial membrane potential and cGAS‐STING activation in CKIP‐1‐silenced group were observed. The involvement of mitochondrial homeostasis‐related TFAM/cGAS‐STING axis in CKIP‐1‐silenced OSCC cells was finally demonstrated by tetramethylpyrazine (TMP) that inhibits TFAM degradation. Taken together, our study demonstrated that CKIP‐1 silencing could significantly antagonize OSCC via TFAM/cGAS‐STING axis, which may provide a candidate target for OSCC treatment. [ABSTRACT FROM AUTHOR]

Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. The full localization of the inner scaffold protein Fam161A requires Poc1. As ciliary forces are increased, Fam161A is reduced, indicative of a force-dependent molecular remodeling of the inner scaffold. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases pose a significant public health challenge globally, necessitating the development of effective treatments to mitigate the risk of cardiovascular diseases. Recently, circular RNAs (circRNAs), a novel class of non-coding RNAs, have been recognized for their role in cardiovascular disease. Aberrant expression of circRNAs is closely linked with changes in various cellular and pathophysiological processes within the cardiovascular system, including metabolism, proliferation, stress response, and cell death. Functionally, circRNAs serve multiple roles, such as acting as a microRNA sponge, providing scaffolds for proteins, and participating in protein translation. Owing to their unique properties, circRNAs may represent a promising biomarker for predicting disease progression and a potential target for cardiovascular drug development. This review comprehensively examines the properties, biogenesis, and potential mechanisms of circRNAs, enhancing understanding of their role in the pathophysiological processes impacting cardiovascular disease. Furthermore, the prospective clinical applications of circRNAs in the diagnosis, prognosis, and treatment of cardiovascular disease are addressed. [ABSTRACT FROM AUTHOR]

Mouse double minute 2 (MDM2) is an oncoprotein that is frequently overexpressed in tumors and enhances cellular transformation. Owing to the important role of MDM2 in modulating p53 function, it is crucial to understand the mechanism underlying the regulation of MDM2 levels. We identified ribosomal protein S4X-linked (RPS4X) as a novel binding partner of MDM2 and showed that RPS4X promotes MDM2 stability. RPS4X suppressed polyubiquitination of MDM2 by suppressing homodimer formation and preventing auto-ubiquitination. Moreover, RPS4X inhibited the interaction between MDM2 and Cullin1, a scaffold protein of the Skp1-Cullin1-F-box protein (SCF) complex and an E3 ubiquitin ligase for MDM2. RPS4X expression in cells enhanced the steady-state level of MDM2 protein. RPS4X was associated not only with MDM2 but also with Cullin1 and then blocked the MDM2/Cullin1 interaction. This is the first report of an interaction between ribosomal proteins (RPs) and Cullin1. Our results contribute to the elucidation of the MDM2 stabilization mechanism in cancer cells, expanding our understanding of the new functions of RPs. [ABSTRACT FROM AUTHOR]

The Motin family proteins (Motins) are a class of scaffolding proteins consisting of Angiomotin (AMOT), AMOT‐like protein 1 (AMOTL1), and AMOT‐like protein 2 (AMOTL2). Motins play a pivotal role in angiogenesis, tumorigenesis, and neurogenesis by modulating multiple cellular signaling pathways. Recent findings indicate that Motins are components of the Hippo pathway, a signaling cascade involved in development and cancer. This review discusses how Motins are integrated into the Hippo signaling network, as either upstream regulators or downstream effectors, to modulate cell proliferation and migration. The repression of YAP/TAZ by Motins contributes to growth inhibition, whereas subcellular localization of Motins and their interactions with actin fibers are critical in regulating cell migration. The net effect of Motins on cell proliferation and migration may contribute to their diverse biological functions. [ABSTRACT FROM AUTHOR]

Protein engineering techniques have vastly expanded their domain of impact, notably following the success of antibodies. Likewise, smaller peptide therapeutics have carved an increasingly significant niche for themselves in the pharmaceutical landscape. The concept of grafting such peptides onto larger protein scaffolds, thus harvesting the advantages of both, has given rise to a variety of protein engineering strategies that are reviewed herein. We also describe our own "Lasso‐Grafting" approach, which combines traditional grafting concepts with mRNA display to streamline the production of multiple grafted drug candidates for virtually any target. [ABSTRACT FROM AUTHOR]

Aim: Striatin (Strn) is a scaffold protein expressed in cardiomyocytes (CMs) and alteration of its expression are described in various cardiac diseases. However, the alteration underlying its pathogenicity have been poorly investigated. Methods: We studied the role(s) of cardiac Strn gene (STRN) by comparing the functional properties of CMs, generated from Strn‐KO and isogenic WT mouse embryonic stem cell lines. Results: The spontaneous beating rate of Strn‐KO CMs was faster than WT cells, and this correlated with a larger fast INa conductance and no changes in If. Paced (2–8 Hz) Strn‐KO CMs showed prolonged action potential (AP) duration in comparison with WT CMs and this was not associated with changes in ICaL and IKr. Motion video tracking analysis highlighted an altered contraction in Strn‐KO CMs; this was associated with a global increase in intracellular Ca2+, caused by an enhanced late Na+ current density (INaL) and a reduced Na+/Ca2+ exchanger (NCX) activity and expression. Immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in Strn‐KO CMs than in WT. Indeed, incubation of Strn‐KO CMs with the microtubule stabilizer taxol, induced a rescue (downregulation) of INa conductance toward WT levels. Conclusion: Loss of STRN alters CMs electrical and contractile profiles and affects cell functionality by a disarrangement of Strn‐related multi‐protein complexes. This leads to impaired microtubules dynamics and Na+ channels trafficking to the plasma membrane, causing a global Na+ and Ca2+ enhancement. [ABSTRACT FROM AUTHOR]

Late endosomal/lysosomal adaptor, MAPK and mTOR, or LAMTOR, is a scaffold protein complex that senses nutrients and integrates growth factor signaling. The role of LAMTOR4 in tumorigenesis is still unknown. However, there is a considerable possibility that LAMTOR4 is directly involved in tumor cell proliferation and metastasis. In the current study, we investigated the protein expression of LAMTOR4 in a cohort of 314 men who were undergoing transurethral resection of prostate (TURP) consisting of incidental, advanced and castration-resistant cases. We also correlated the data with ERG and PTEN genomic status and clinicopathological features including Gleason score and patients' outcome. Additionally, we performed in vitro experiments utilizing knockdown of LAMTOR4 in prostate cell lines, and we performed mRNA expression assessment using TCGA prostate adenocarcinoma (TCGA-PRAD) to explore the potential differentially expressed genes and pathways associated with LAMTOR4 overexpression in PCa patients. Our data indicate that high LAMTOR4 protein expression was significantly associated with poor overall survival (OS) (HR: 1.44, CI: 1.01–2.05, p = 0.047) and unfavorable cause-specific survival (CSS) (HR: 1.71, CI: 1.06–2.77, p = 0.028). Additionally, when high LAMTOR4 expression was combined with PTEN-negative cases (score 0), we found significantly poorer OS (HR: 2.22, CI: 1.37–3.59, p = 0.001) and CSS (HR: 3.46, CI: 1.86–6.46, p < 0.0001). Furthermore, ERG-positive cases with high LAMTOR4 exhibited lower OS (HR: 1.98, CI: 1.18–3.31, p = 0.01) and CSS (HR: 2.54, CI: 1.32–4.87, p = 0.005). In vitro assessment showed that knockdown of LAMTOR4 decreases PCa cell proliferation, migration, and invasion. Our data further showed that knockdown of LAMTOR4 in the LNCaP cell line significantly dysregulated the β catenin/mTOR pathway and tumorigenesis associated pathways. Inhibiting components of the mTOR pathway, including LAMTOR4, might offer a strategy to inhibit tumor progression and metastasis in prostate cancer. [ABSTRACT FROM AUTHOR]

Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling. [ABSTRACT FROM AUTHOR]

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein–protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins. [ABSTRACT FROM AUTHOR]

Itaconic acid is an excellent polymeric precursor with a wide range of industrial applications. The efficient production of itaconate from various renewable substrates was demonstrated by engineered Escherichia coli. However, limitation in the itaconic acid precursor supply was revealed by finding out the key intermediate of the tricarboxylic acid in the itaconic acid pathway. Efforts of enhancing the cis-aconitate flux and preserving the isocitrate pool to increase itaconic acid productivity are required. In this study, we introduce a synthetic protein scaffold system between CadA and AcnA to physically combine the two enzymes. Through the introduction of a synthetic protein scaffold, 2.1 g L−1 of itaconic acid was produced at pH 7 and 37 °C. By fermentation, 20.1 g L−1 for 48 h of itaconic acid was produced with a yield of 0.34 g g−1 glycerol. These results suggest that carbon flux was successfully increased itaconic acid productivity. [ABSTRACT FROM AUTHOR]

A multifunctional scaffold protein termed Disabled-2 (Dab2) has recently gained attention in the scientific community and has emerged as a promising candidate in the realm of cancer research. Dab2 protein is involved in a variety of signaling pathways, due to which its significance in the pathogenesis of several carcinomas has drawn considerable attention. Dab2 is essential for controlling the advancement of cancer because it engages in essential signaling pathways such as the Wnt/β-catenin, epidermal growth factor receptor (EGFR), and transforming growth factor-beta (TGF-β) pathways. Dab2 can also repress epithelial-mesenchymal transition (EMT) which is involved in tumor progression with metastatic expansion and adds another layer of significance to its possible impact on cancer spread. Furthermore, the role of Dab2 in processes such as cell growth, differentiation, apoptosis, invasion, and metastasis has been explored in certain investigative studies suggesting its significance. The present review examines the role of Dab2 in the pathogenesis of various cancer subtypes including breast cancer, ovarian cancer, gastric cancer, prostate cancer, and bladder urothelial carcinoma and also sheds some light on its potential to act as a therapeutic target and a prognostic marker in the treatment of various carcinomas. By deciphering this protein's diverse signaling, we hope to provide useful insights that may pave the way for novel therapeutic techniques and tailored treatment approaches in cancer management. Preclinical and clinical trial data on the impact of Dab2 regulation in cancer have also been included, allowing us to delineate role of Dab2 in tumor suppressor function, as well as its correlation with disease stage classification and potential therapy options. However, we observed that there is very scarce data in the form of studies on the evaluation of Dab2 role and treatment function in carcinomas, and further research into this matter could prove beneficial in the generation of novel therapeutic agents for patient-centric and tailored therapy, as well as early prognosis of carcinomas. [ABSTRACT FROM AUTHOR]

Affilin proteins, artificial binding proteins based on the ubiquitin scaffold, have been generated by directed protein evolution to yield de-novo variants that bind the extra-domain B (EDB) of oncofetal fibronectin, an established marker of tumor neovasculature. The crystal structures of two EDB-specific Affilin variants reveal a striking structural plasticity of the ubiquitin scaffold, characterised by β-strand slippage, leading to different negative register shifts of the β5 strands. This process recruits amino acid residues from β5 towards the N-terminus to an adjacent loop region and subsequent residues into β5, respectively, remodeling the binding interface and leading to target specificity and affinity. Protein backbone alterations resulting from β-strand register shifts, as seen in the ubiquitin fold, can pose additional challenges to protein engineering as structural evidence of these events is still limited and they are difficult to predict. However, they can surface under the selection pressure of directed evolution and suggest that backbone plasticity allowing β-strand slippages can increase structural diversity, enhancing the evolutionary potential of a protein scaffold. The authors show that diubiquitin-derived artificial binding proteins (Affilin molecules) targeting oncofetal fibronectin employ β-strand register shifts in the scaffold, remodelling the binding interface to confer target affinity. [ABSTRACT FROM AUTHOR]

Introduction: Lipopolysaccharide-responsive and beige-liκe anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PκA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells. Aim: To compare and evaluate spleen-derived B cell responses to BCR crosslinκing in C57BL6 Lrba-/- and Lrba+/+ mice. Materials and methods: Spleen-derived B cells were obtained from 8 to 12-weeκ-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinκing was assessed by the F(ab')2 anti-m chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot. Results: Lrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinκing in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBa, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinκing in Lrba-/- B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected. Discussion: These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter. [ABSTRACT FROM AUTHOR]

During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that β-arrestin1 and β-arrestin2 (β-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that β-arrestins are expressed in rat commissural neurons. We also found that Smo, β-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on β-arrestins. β-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that β-arrestins are required to activate Src kinase downstream of Shh. β-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative β-arrestins, β-arrestin1 V53D which blocks the internalization of Smo and β-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative β-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that β-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance. [ABSTRACT FROM AUTHOR]

The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization. [ABSTRACT FROM AUTHOR]

Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoeshaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on a-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindleshaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK; encoded by PTK2) was discovered over 30 years ago as a cytoplasmic protein tyrosine kinase that is localized to cell adhesion sites, where it is activated by integrin receptor binding to extracellular matrix proteins. FAK is ubiquitously expressed and functions as a signaling scaffold for a variety of proteins at adhesions and in the cell cytoplasm, and with transcription factors in the nucleus. FAK expression and intrinsic activity are essential for mouse development, with molecular connections to cell motility, cell survival and gene expression. Notably, elevated FAK tyrosine phosphorylation is common in tumors, including pancreatic and ovarian cancers, where it is associated with decreased survival. Small molecule and orally available FAK inhibitors show on-target inhibition in tumor and stromal cells with effects on chemotherapy resistance, stromal fibrosis and tumor microenvironment immune function. Herein, we discuss recent insights regarding mechanisms of FAK activation and signaling, its roles as a cytoplasmic and nuclear scaffold, and the tumor-intrinsic and -extrinsic effects of FAK inhibitors. We also discuss results from ongoing and advanced clinical trials targeting FAK in low- and high-grade serous ovarian cancers, where FAK acts as a master regulator of drug resistance. Although FAK is not known to be mutationally activated, preventing FAK activity has revealed multiple tumor vulnerabilities that support expanding clinical combinatorial targeting possibilities. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1–400 aa), Linker (246–446 aa) and Adh (444–687 aa) to evaluate their role in virulence. The TrophozBro11–400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226–446 also increased the virulence properties, but with lower effect than the TrophozBro11–400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444–687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes. [ABSTRACT FROM AUTHOR]

SUMMARY: The RNA regulatory network is a complex and dynamic regulation in plant cells involved in mRNA modification, translation, and degradation. Ras‐GAP SH3 domain‐binding protein (G3BP) is a scaffold protein for the assembly of stress granules (SGs) and is considered an antiviral component in mammals. However, the function of G3BP during virus infection in plants is still largely unknown. In this study, four members of the G3BP‐like proteins (NtG3BPLs) were identified in Nicotiana tabacum and the expression levels of NtG3BPL1 were upregulated during chilli veinal mottle virus (ChiVMV) infection. NtG3BPL1 was localized in the nucleus and cytoplasm, forming cytoplasmic granules under transient high‐temperature treatment, whereas the abundance of cytoplasmic granules was decreased under ChiVMV infection. Overexpression of NtG3BPL1 inhibited ChiVMV infection and delayed the onset of symptoms, whereas knockout of NtG3BPL1 promoted ChiVMV infection. In addition, NtG3BPL1 directly interacted with ChiVMV 6K2 protein, whereas 6K2 protein had no effect on NtG3BPL1‐derived cytoplasmic granules. Further studies revealed that the expression of NtG3BPL1 reduced the chloroplast localization of 6K2‐GFP and the NtG3BPL1‐6K2 interaction complex was localized in the cytoplasm. Furthermore, NtG3BPL1 promoted the degradation of 6K2 through autophagy pathway, and the accumulation of 6K2 and ChiVMV was affected by autophagy activation or inhibition in plants. Taken together, our results demonstrate that NtG3BPL1 plays a positive role in tobacco resistance against ChiVMV infection, revealing a novel mechanism of plant G3BP in antiviral strategy. Significance Statement: G3BP is a key component of cytoplasmic stress granules and exerts antiviral effects in mammals, but its function during plant virus infection remains unclear. Here, we revealed that NtG3BPL1 is induced upon ChiVMV infection and interacts with ChiVMV 6K2 to disturb the chloroplast localization of 6K2, and promote the degradation of 6K2, thereby limiting ChiVMV infection. This study enhances our understanding of the function of G3BP in plant virus infection. [ABSTRACT FROM AUTHOR]

This article comments on: Li Z, Zhang D, Liang X, Liang J. 2024. Receptor for Activated C Kinase 1 counteracts ABSCISIC ACID INSENSITIVE5-mediated inhibition of seed germination and post-germinative growth in Arabidopsis. Journal of Experimental Botany 75, 3932–3945. [ABSTRACT FROM AUTHOR]

The prohibitins Phb1 and Phb2 assemble at the mitochondrial inner membrane to form a multi-dimeric complex. These scaffold proteins are highly conserved in eukaryotic cells, from yeast to mammals, and have been implicated in a variety of mitochondrial functions including aging, proliferation, and degenerative and metabolic diseases. In mammals, PHB2 regulates PINK1-PRKN mediated mitophagy by interacting with lipidated MAP1LC3B/LC3B. Despite their high conservation, prohibitins have not been linked to mitophagy in budding yeasts. In this study, we demonstrate that both Phb1 and Phb2 are required to sustain mitophagy in Saccharomyces cerevisiae. Prohibitin-dependent mitophagy requires formation of the Phb1-Phb2 complex and a conserved AIM/LIR-like motif identified in both yeast prohibitins. Furthermore, both Phb1 and Phb2 interact and exhibit mitochondrial colocalization with Atg8. Interestingly, we detected a basal C terminus processing of the mitophagy receptor Atg32 that depends on the presence of the i-AAA Yme1. In the absence of prohibitins this processing is highly enhanced but reverted by the inactivation of the rhomboid protease Pcp1. Together our results revealed a novel role of yeast prohibitins in mitophagy through its interaction with Atg8 and regulating an Atg32 proteolytic event. Abbreviation: AIM/LIR: Atg8-family interacting motif/LC3-interacting region; ANOVA: analysis of variance; ATG/Atg: autophagy related; C terminus/C-terminal: carboxyl terminus/carboxyl-terminal; GFP: green fluorescent protein; HA: human influenza hemagglutinin; Idh1: isocitrate dehydrogenase 1; MAP1C3B/LC3B: microtubule associated protein 1 light chain 3 beta; mCh: mCherry; MIM: mitochondrial inner membrane; MOM: mitochondrial outer membrane; N starvation: nitrogen starvation; N terminus: amino terminus; PARL: presenilin associated rhomboid like; Pcp1: processing of cytochrome c peroxidase 1; PCR: polymerase chain reaction; PGAM5: PGAM family member 5 mitochondrial serine/threonine protein phosphatase; PHBs/Phb: prohibitins; PINK1: PTEN induced kinase 1; PMSF: phenylmethylsulfonyl fluoride; PRKN: parkin RBR E3 ubiquitin protein ligase; SD: synthetic defined medium; SDS: sodium dodecyl sulfate; SMD-N: synthetic defined medium lacking nitrogen; WB: western blot; WT: wild type; Yme1: yeast mitochondrial escape 1; YPD: yeast extract-peptone-dextrose medium; YPLac: yeast extract-peptone-lactate medium. [ABSTRACT FROM AUTHOR]

Nucleotide excision repair (NER) is the most universal repair pathway, which removes a wide range of DNA helix-distorting lesions caused by chemical or physical agents. The final steps of this repair process are gap-filling repair synthesis and subsequent ligation. XPA is the central NER scaffolding protein factor and can be involved in post-incision NER stages. Replication machinery is loaded after the first incision of the damaged strand that is performed by the XPF–ERCC1 nuclease forming a damaged 5′-flap processed by the XPG endonuclease. Flap endonuclease I (FEN1) is a critical component of replication machinery and is absolutely indispensable for the maturation of newly synthesized strands. FEN1 also contributes to the long-patch pathway of base excision repair. Here, we use a set of DNA substrates containing a fluorescently labeled 5′-flap and different size gap to analyze possible repair factor–replication factor interactions. Ternary XPA–FEN1–DNA complexes with each tested DNA are detected. Furthermore, we demonstrate XPA–FEN1 complex formation in the absence of DNA due to protein–protein interaction. Functional assays reveal that XPA moderately inhibits FEN1 catalytic activity. Using fluorescently labeled XPA, formation of ternary RPA–XPA–FEN1 complex, where XPA accommodates FEN1 and RPA contacts simultaneously, can be proposed. We discuss possible functional roles of the XPA–FEN1 interaction in NER related DNA resynthesis and/or other DNA metabolic processes where XPA can be involved in the complex with FEN1. [ABSTRACT FROM AUTHOR]

Summary: Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis.Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free‐living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate.Using Y2H library screening, BiFC and Co‐IP, we demonstrated an interaction between the mitogen‐activated protein kinase LbSakA and LbNoxR. LbSakA‐mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis‐related genes involved in dampening host defense reactions.These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks. [ABSTRACT FROM AUTHOR]

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites. [ABSTRACT FROM AUTHOR]

Background: Acetaminophen (APAP) is commonly used as an antipyretic and analgesic agent. Excessive APAP can induce liver toxicity, known as APAP-induced liver injury (ALI). The metabolism and pathogenesis of APAP have been extensively studied in recent years, and many cellular processes such as autophagy, mitochondrial oxidative stress, mitochondrial dysfunction, and liver regeneration have been identified to be involved in the pathogenesis of ALI. Caveolin-1 (CAV-1) as a scaffold protein has also been shown to be involved in the development of various diseases, especially liver disease and tumorigenesis. The role of CAV-1 in the development of liver disease and the association between them remains a challenging and uncharted territory. Summary: In this review, we briefly explore the potential therapeutic effects of CAV-1 on ALI through autophagy, oxidative stress, and lipid metabolism. Further research to better understand the mechanisms by which CAV-1 regulates liver injury will not only enhance our understanding of this important cellular process, but also help develop new therapies for human disease by targeting CAV-1 targets. Key Messages: This review briefly summarizes the potential protective mechanisms of CAV-1 against liver injury caused by APAP. [ABSTRACT FROM AUTHOR]

During mitosis, the Bub1--Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1--Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1--BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB- 1--BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1--Bub3 localization and functions may be conserved. [ABSTRACT FROM AUTHOR]

Generating active, pure, and monodisperse protein remains a major bottleneck for structural studies using X‐ray crystallography and cryo‐electron microscopy (cryo‐EM). The current methodology heavily relies on overexpressing the recombinant protein fused with a histidine tag in conventional expression systems and evaluating the quality and stability of purified protein using size exclusion chromatography (SEC). This requires a large amount of protein and can be highly laborious and time consuming. Therefore, this approach is not suitable for high‐throughput screening and low‐expressing macromolecules, particularly eukaryotic membrane proteins. Using fluorescent proteins fused to the target protein (applicable to both soluble and membrane proteins) enables rapid and efficient screening of expression level and monodispersity of tens of unpurified constructs using fluorescence‐based size exclusion chromatography (FSEC). Moreover, FSEC proves valuable for screening multiple detergents to identify the most stabilizing agent in the case of membrane proteins. Additionally, FSEC can facilitate nanodisc reconstitution by determining the optimal ratio of membrane scaffold protein (MSP), lipids, and target protein. The distinct advantages offered by FSEC indicate that fluorescent proteins can serve as a viable alternative to commonly used affinity tags for both characterization and purification purposes. In this review, I will summarize the advantages of this technique using examples from my own work. It should be noted that this article is not intended to provide an exhaustive review of all available literature, but rather to offer representative examples of FSEC applications. [ABSTRACT FROM AUTHOR]

During epithelial morphogenesis, the apical junctions connecting cells must remodel as cells change shape and make new connections with their neighbors. In the C. elegans embryo, new apical junctions form when epidermal cells migrate and seal with one another to encase the embryo in skin ('ventral enclosure'), and junctions remodel when epidermal cells change shape to squeeze the embryo into a worm shape ('elongation'). The junctional cadherin-catenin complex (CCC), which links epithelial cells to each other and to cortical actomyosin, is essential for C. elegans epidermal morphogenesis. RNAi genetic enhancement screens have identified several genes encoding proteins that interact with the CCC to promote epidermal morphogenesis, including the scaffolding protein Afadin (AFD-1), whose depletion alone results in only minor morphogenesis defects. Here, by creating a null mutation in afd-1 , we show that afd-1 provides a significant contribution to ventral enclosure and elongation on its own. Unexpectedly, we find that afd-1 mutant phenotypes are strongly modified by diet, revealing a previously unappreciated parental nutritional input to morphogenesis. We identify functional interactions between AFD-1 and the CCC by demonstrating that E-cadherin is required for the polarized distribution of AFD-1 to cell contact sites in early embryos. Finally, we show that afd-1 promotes the enrichment of polarity regulator, and CCC-interacting protein, PAC-1/ARHGAP21 to cell contact sites, and we identify genetic interactions suggesting that afd-1 and pac-1 regulate epidermal morphogenesis at least in part through parallel mechanisms. Our findings reveal that C. elegans AFD-1 makes a significant contribution to epidermal morphogenesis and functionally interfaces with core and associated CCC proteins. [Display omitted] • afd-1 null mutant reveals diet-influenced roles for Afadin in C. elegans epidermal morphogenesis. • afd-1 genetically interacts with RhoGAP pac-1 to regulate epidermal morphogenesis. • E-cadherin enriches AFD-1 at cell contacts. • AFD-1 promotes PAC-1 cell localization. [ABSTRACT FROM AUTHOR]

Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha‐synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune‐specific response against alpha‐synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha‐synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha‐synuclein and its amyloid form. The prion domain of the fungal HET‐s protein was selected as a scaffold to introduce select epitopes from the surface of alpha‐synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in‐register beta‐sheet structure seen in alpha‐synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha‐synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha‐synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha‐synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha‐synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha‐synuclein, provides a promising avenue toward developing effective vaccines against alpha‐synuclein fibrils. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are emerging as promising carriers for the delivery of therapeutic biologics. Genetic engineering represents a robust strategy for loading proteins of interest into EVs. Identification of EV‐enriched proteins facilitates protein cargo loading efficiency. Many EV‐enriched proteins are sorted into EVs via an endosomal sorting complex required for transport (ESCRT)‐dependent pathway. In parallel, viruses hijack this EV biosynthesis machinery via conserved late domain motifs to promote egress from host cells. Inspired by the similarity of biogenesis between EVs and viruses, we developed a synthetic, Late domain‐based EV scaffold protein that enables the display of a set of single chain variable fragments (scFvs) on the EV surface. We named this scaffold the Late domain‐based exosomal antibody surface display platform (LEAP). We applied the LEAP scaffold to reprogramme HEK293T cell‐derived EVs to elicit T‐cell anti‐tumor immunity by simultaneously displaying αPD‐L1 and αCD3 scFvs on the EV surface (denoted as αPD‐L1×αCD3 bispecific T‐cell engaging exosomes, BiTExos). We demonstrated that αPD‐L1×αCD3 BiTExos actively redirected T cells to bind to PD‐L1+ tumor cells, promoting T‐cell activation, proliferation and tumoricidal cytokine production. Furthermore, the αPD‐L1×αCD3 BiTExos promoted T‐cell infiltration into the tumor microenvironment to mitigate the tumor burden in vivo. Our study suggested that the LEAP scaffold may serve as a platform for EV surface display and could be applied for a broad range of EV‐based biomedical applications. [ABSTRACT FROM AUTHOR]

Filamentous temperature-sensitive Z (FtsZ) is a tubulin-like GTPase that is highly conserved in bacteria and plants. It polymerizes into a ring at the division site of bacteria and chloroplasts and serves as the scaffold protein of the division complex. While a single FtsZ is present in bacteria and cyanobacteria, there are two subfamilies, FtsZ1 and FtsZ2 in the green lineage, and FtsZA and FtsZB in red algae. In Arabidopsis thaliana , the C-terminal motifs of AtFtsZ1 (Z1C) and AtFtsZ2-1 (Z2C) display distinct functions in the regulation of chloroplast division. Z1C exhibits weak membrane-binding activity, whereas Z2C engages in the interaction with the membrane protein AtARC6. Here, we provide evidence revealing the distinct traits of the C-terminal motifs of FtsZ1 and FtsZ2 throughout the plant evolutionary process. In a range of plant species, the C-terminal motifs of FtsZ1 exhibit diverse membrane-binding properties critical for regulating chloroplast division. In chlorophytes, the C-terminal motifs of FtsZ1 and FtsZ2 exhibit both membrane-binding and protein interaction functions, which are similar to those of cyanobacterial FtsZ and red algal FtsZA. During the transition from algae to land plants, the functions of the C-terminal motifs of FtsZ1 and FtsZ2 exhibit differentiation. FtsZ1 lost the function of interacting with ARC6 in land plants, and the membrane-binding activity of FtsZ2 was lost in ferns. Our findings reveal the functional differentiation of the C-terminal motifs of FtsZs during plant evolution, which is critical for chloroplast division. [ABSTRACT FROM AUTHOR]

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection. The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. Here, Ukleja et al. show that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability under cellular stress and during infection. [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation (LLPS), an emerging biophysical phenomenon, can sequester molecules to implement physiological and pathological functions. LLPS implements the assembly of numerous membraneless chambers, including stress granules and P‐bodies, containing RNA and protein. RNA–RNA and RNA–protein interactions play a critical role in LLPS. Scaffolding proteins, through multivalent interactions and external factors, support protein–RNA interaction networks to form condensates involved in a variety of diseases, particularly neurodegenerative diseases and cancer. Modulating LLPS phenomenon in multiple pathogenic proteins for the treatment of neurodegenerative diseases and cancer could present a promising direction, though recent advances in this area are limited. Here, we summarize in detail the complexity of LLPS in constructing signaling pathways and highlight the role of LLPS in neurodegenerative diseases and cancers. We also explore RNA modifications on LLPS to alter diseases progression because these modifications can influence LLPS of certain proteins or the formation of stress granules, and discuss the possibility of proper manipulation of LLPS process to restore cellular homeostasis or develop therapeutic drugs for the eradication of diseases. This review attempts to discuss potential therapeutic opportunities by elaborating on the connection between LLPS, RNA modification, and their roles in diseases. [ABSTRACT FROM AUTHOR]