Exogenous phosphatidylserine (PS) is cytotoxic to Chinese hamster ovary (CHO) cells with a PS-resistant CHO cell mutant exhibiting impaired transbilayer movement of a fluorescent PS analog at the plasma membrane. Here, w...Exogenous phosphatidylserine (PS) is cytotoxic to Chinese hamster ovary (CHO) cells with a PS-resistant CHO cell mutant exhibiting impaired transbilayer movement of a fluorescent PS analog at the plasma membrane. Here, we demonstrate that both mRNA and protein levels of 14-3-3 zeta are markedly reduced in this mutant, while knockdown of 14-3-3 zeta in wild-type CHO cells confers partial resistance to exogenous PS. Consistently, these knockdown cells display defective transbilayer movement of 1-palmitoyl-2-{6-[(7-nitro-1,3-benz-2-oxadiazol-4-yl) amino] hexanoyl}-sn-glycero-3-phosphoserine (C-NBD-PS). However, 14-3-3 zeta knockdown does not further enhance PS resistance in ATP11C-deficient cells. These findings indicate that 14-3-3 zeta plays a regulatory role in ATP11C-dependent transbilayer movement of PS at the plasma membrane. Impact statement 14-3-3ζ regulates ATP11C-dependent phosphatidylserine (PS) transbilayer movement at the plasma membrane. Reduced 14-3-3ζ impairs PS flipping and confers resistance to exogenous PS in CHO cells, identifying 14-3-3ζ as a key modulator of phospholipid asymmetry and lipid-induced cytotoxicity.
Reversible conformational dynamics of membrane proteins are essential for intracellular signaling, but no method enables their irreversible arrest in living cells. Here, we developed a genetically encoded proximity-based...Reversible conformational dynamics of membrane proteins are essential for intracellular signaling, but no method enables their irreversible arrest in living cells. Here, we developed a genetically encoded proximity-based lockdown enzyme derived from an engineered transglutaminase catalytic core (TGC) that covalently crosslinks membrane proteins. By fusing TGC to the endoplasmic reticulum (ER)-resident microprotein ALN encoded by a short open reading frame (sORF), we created an organelle-specific module that selectively catalyzes covalent crosslinking within the SERCA Ca pump, strongly suppressing its ATP-dependent pump activity and arresting ER Ca signaling. This engineered lockdown enzyme remodels ER membrane protein architecture and restricts conformational dynamics, providing a versatile platform for long-term covalent control of intracellular signaling and a foundation for future therapeutic cellular applications. Impact statement Our proximity-based lockdown enzyme, engineered from microbial transglutaminase, provides a new strategy to covalently arrest the conformational states of organelle-resident membrane proteins in living cells, enabling long-term control of intracellular signaling and establishing a foundation for next-generation cellular therapeutics.
UBR5 is a HECT-type E3 ubiquitin ligase that assembles K48-linked ubiquitin chains and generates K48-linked branched chains. UBR4 is another E3 that forms K48-linked chains through an atypical hemi-RING-like domain. To d...UBR5 is a HECT-type E3 ubiquitin ligase that assembles K48-linked ubiquitin chains and generates K48-linked branched chains. UBR4 is another E3 that forms K48-linked chains through an atypical hemi-RING-like domain. To define substrates specific to each ligase, we analyzed tandem mass tag proteomics data and identified candidates that accumulated after UBR5 or UBR4 knockdown, respectively. UBR5 depletion caused a marked delay in turnover of the chromatin regulator ATAD2. We found that ATAD2 associated with UBR5 in co-immunoprecipitation assays, and UBR5 promoted ubiquitylation of ATAD2 in vitro and in cells. RNA-sequencing further showed that the expression of cell cycle-related genes was antagonistically regulated by ATAD2 and UBR5. These findings identify ATAD2 as a UBR5 substrate and reveal a regulatory module controlling gene expression.
Linear (Met1-linked) polyubiquitin chains play essential roles in NF-κB signaling, with readers such as A20 specifically recognizing these chains via specialized domains. Although structural data exist for the linear diu...Linear (Met1-linked) polyubiquitin chains play essential roles in NF-κB signaling, with readers such as A20 specifically recognizing these chains via specialized domains. Although structural data exist for the linear diubiquitin-A20 ZF7 complex, the basis for its strong preference for linear polyubiquitin remains unclear. Here, we investigated the early steps of ubiquitin recognition by A20-ZF7, identifying that A20-ZF7 exhibits weak binding to monoubiquitin and distinct exchange kinetics at its two ubiquitin-binding sites, with slow exchange at the proximal site reflecting stronger binding. Comparison of monoubiquitin and linear diubiquitin binding suggested a multistep mechanism involving a kinetically resolvable intermediate state absent in the monoubiquitin interaction. This intermediate likely facilitates proper ubiquitin chain positioning and contributes to A20-ZF7's specificity for linear polyubiquitin. Impact statement We reveal transient intermediate states in ubiquitin recognition by A20 ZF7, showing that specificity for linear chains arises from a multistep kinetic pathway. This work highlights the importance of dynamic binding processes in decoding ubiquitin signaling.
Pulmonary neuroendocrine cell (PNEC) hyperplasia often occurs in lung diseases, including allergic asthma. We previously reported that PNEC-derived calcitonin gene-related peptide (CGRP) likely stimulates group 2 innate...Pulmonary neuroendocrine cell (PNEC) hyperplasia often occurs in lung diseases, including allergic asthma. We previously reported that PNEC-derived calcitonin gene-related peptide (CGRP) likely stimulates group 2 innate lymphoid cells (ILC2), exacerbating asthma phenotypes in a mouse model. Here, we investigate the role of glial cell-line derived neurotrophic factor (GDNF) and rearranged during transfection (RET) signaling in PNEC hyperplasia and its therapeutic potential in asthma. PNECs expressed GDNF receptors, which were activated primarily by infiltrating inflammatory cells. Application of a RET-specific inhibitor suppressed ILC2 levels, PNEC hyperplasia and airway allergic responses. We suggest that GDNF-RET signaling promotes PNEC hyperplasia and that the PNEC-CGRP-ILC2 axis is closely associated with the development of allergic asthma, presenting a possible new treatment strategy. Impact statement Our study is the first to indicate the possibility of controlling pulmonary neuroendocrine cell (PNEC) hyperplasia and acute allergic airway inflammation through RET signaling, which could lead to elucidating the mechanism underlying the PNEC hyperplasia-immune relationship in asthma. We propose that targeting this could be a new treatment strategy.
Plasmodium falciparum exports proteins into host erythrocytes for survival, but the roles of many of these proteins remain unexplored. Here, we used recombinant protein constructs and antibodies corresponding to Plasmodi...Plasmodium falciparum exports proteins into host erythrocytes for survival, but the roles of many of these proteins remain unexplored. Here, we used recombinant protein constructs and antibodies corresponding to Plasmodium falciparum ring-infected erythrocyte surface antigen-3 (PfRESA3) to identify it as a dense granule merozoite protein exported early to the inner erythrocyte membrane, where it associates with the cytoskeleton. Constructs lacking the J domain bound inside-out vesicles, implicating PRESAN and DnaJ-X in cytoskeletal interactions. Recombinant PfRESA3 constructs stimulated the ATPase activity of human HsHSPA8 and enhanced malate dehydrogenase (MDH) refolding. However, truncated PfRESA3 constructs did not significantly enhance MDH refolding by HsHSPA8. These findings suggest PfRESA3 modulates host HsHSPA8 to support cytoskeletal remodeling/parasite protein folding early in infection. Impact statement Our study uncovers a high-stakes hijacking of the human erythrocyte by Plasmodium falciparum. By identifying PfRESA3 as a potential co-chaperone that anchors to the host's cytoskeleton and recruits human chaperones, this study reveals a sophisticated survival strategy. It exposes a critical vulnerability in the parasite's early development inside the human erythrocyte.
While the downstream effectors of the hyperosmotic stress response are relatively well characterized, the primary molecular sensors responsible for initial stress detection remain poorly defined. In this study, we demons...While the downstream effectors of the hyperosmotic stress response are relatively well characterized, the primary molecular sensors responsible for initial stress detection remain poorly defined. In this study, we demonstrate that hyperosmotic stress triggers a rapid and transient mono(ADP-ribosyl)ation (MARylation). Beside MARylation, signs of acute genotoxicity are missing and CHK1 activation is observed only upon recovery from osmotic stress. Our data indicate that PARP1 catalyzes its own MARylation in an HPF1 co-factor dependent manner. Biochemical assays further demonstrate that the mono-ADP-ribose moiety is resistant to hydroxylamine treatment, which is a feature of HPF1-directed O-glycosidic bonds. Together, these findings support a model in which PARP1 acts as a sensor of chromatin structure changes induced by hyperosmotic stress leading to its autoMARylation.
Primary mitochondrial disorders (PMDs) are inherited metabolic diseases that most often present with neurological symptoms in infancy or adolescence, underscoring the central importance of mitochondrial function to brain...Primary mitochondrial disorders (PMDs) are inherited metabolic diseases that most often present with neurological symptoms in infancy or adolescence, underscoring the central importance of mitochondrial function to brain health. Historically, the field has emphasized neurodegeneration-consistent with the high energetic demands of postmitotic neurons. However, neurodevelopmental manifestations are now recognized as common early phenotypes, frequently preceding clinical regression in many PMDs. Given the pivotal role of mitochondria in neural stem/progenitor cell maintenance and cell fate decisions, defects in the respiratory chain are poised to disrupt neurogenesis and gliogenesis. Evidence for such developmental vulnerabilities is reviewed here. Likewise, because mitochondrial metabolism and dynamics shift across the oligodendrocyte lineage-from oligodendrocyte precursor cell expansion to differentiation and the energetically intensive phase of myelin synthesis-callosal atrophy in mitochondrial leukoencephalopathies may, at least in part, reflect developmental shortcomings in oligodendrogenesis and myelination. This possibility warrants focused investigation in cellular and in vivo models.
Circadian rhythms are biological cycles of approximately 24 h that align physiology and behavior with the solar day, helping organisms coordinate their functions with the light/dark cycle. These rhythms are generated by...Circadian rhythms are biological cycles of approximately 24 h that align physiology and behavior with the solar day, helping organisms coordinate their functions with the light/dark cycle. These rhythms are generated by molecular circadian clocks found in cells that are composed of transcription/translation negative feedback loops and regulate gene activity and protein production. The field of chemical biology has generated tools to track, modify, and manipulate clock proteins in living systems, providing a meaningful way to study these clocks and their components. Small molecules, covalent tags, and detectable reporters, among others, have been used to reveal how clocks keep time, respond to environmental signals, and differ across organisms. In this review, we highlight and describe chemical biology approaches used to study and modulate molecular circadian mechanisms that have expanded understanding of circadian protein dynamics and interactions in the contexts of mammalian and Drosophila models. The application of chemical biology strategies to study and target circadian clocks and their components can expand our fundamental knowledge via means that are otherwise inaccessible and point toward new strategies for treating clock-related disorders.
Terpene synthases (TPSs) generate complex hydrocarbon scaffolds through carbocationic cyclization cascades that demand precise active-site control to stabilize reactive intermediates. While π-cation and electrostatic int...Terpene synthases (TPSs) generate complex hydrocarbon scaffolds through carbocationic cyclization cascades that demand precise active-site control to stabilize reactive intermediates. While π-cation and electrostatic interactions are established stabilizing factors, the role of methionine has remained unclear. Here, we identify a methionine-rich active site in hydropyrene synthase (HpS), a bacterial Class I TPS involved in pseudopterosin biosynthesis. Crystallography, mutagenesis, and multiscale QM/MM simulations reveal that methionine residues provide steric guidance and direct sulfur-carbocation stabilization during catalysis. Mutations alter product distributions, confirming functional relevance. Quantum chemical calculations indicate that sulfur-carbocation interactions are energetically comparable to π-carbocation interactions. These results uncover a previously unrecognized mechanism of carbocation stabilization in terpene biosynthesis.
Fused in sarcoma (FUS) forms phase-separated condensates implicated in amyotrophic lateral sclerosis (ALS). Although millimolar ATP concentrations paradoxically dissolve FUS condensates through hydrotropic activity, cond...Fused in sarcoma (FUS) forms phase-separated condensates implicated in amyotrophic lateral sclerosis (ALS). Although millimolar ATP concentrations paradoxically dissolve FUS condensates through hydrotropic activity, condensates nevertheless persist in cells, suggesting active regulatory mechanisms. Here, using a reconstituted system, we show that the AAA+ATPase valosin-containing protein (VCP) counteracts ATP-driven dissolution of FUS condensates. VCP preserved both wild-type and ALS-linked P525L condensates under high ATP conditions, and this protection required catalytic ATPase activity rather than stable partitioning into condensates. The effect was abolished by the D2-specific inhibitor ML240. Our findings establish direct biochemical evidence that VCP ATPase activity maintains FUS condensates under high ATP conditions, highlighting ATPase-driven enzymatic control of liquid-liquid phase separation as a potential general principle with implications for neurodegeneration.
The asymmetry of the phospholipids in the cell membrane is fundamental to maintaining normal cellular physiological functions. Phosphatidylserine (PS), a key phospholipid, is typically restricted to the inner side of the...The asymmetry of the phospholipids in the cell membrane is fundamental to maintaining normal cellular physiological functions. Phosphatidylserine (PS), a key phospholipid, is typically restricted to the inner side of the plasma membrane. However, during specific physiological or pathological processes such as apoptosis, PS rapidly flips to the cell surface, serving as an 'eat me' signal that mediates apoptotic cells' recognition. This process is catalyzed by a class of membrane proteins known as 'scramblases'. The Xk-related (Xkr) protein family, particularly Xkr8, has been identified as the main scramblase during apoptosis. As research has progressed in recent years, the functions of other Xkr family members (such as Xkr4 and Xkr9) have gradually come to light. They not only participate in apoptosis, but also play vital roles in various life processes, including nervous system development, auditory formation and tumor immunity. This article systematically reviews the discovery, molecular structure, activation mechanisms, and current research on the Xkr protein family. We also discuss future research directions, aiming to provide a comprehensive understanding of the functional diversity and regulatory networks of this emerging protein family.
CFIm25, a key component of the cleavage factor Im (CFIm) complex needed for mRNA 3' end processing, shows increased protein expression during monocyte-to-macrophage differentiation despite stable mRNA levels. We demonstr...CFIm25, a key component of the cleavage factor Im (CFIm) complex needed for mRNA 3' end processing, shows increased protein expression during monocyte-to-macrophage differentiation despite stable mRNA levels. We demonstrate that poly(C)-binding protein 1 (PCBP1) suppresses CFIm25 translation in monocytes by binding to its long 3' untranslated region (UTR). During differentiation, alternative polyadenylation generates a shorter CFIm25 3'UTR lacking PCBP1 binding sites. RNA immunoprecipitation confirms PCBP1 binding to the long 3'UTR, while ribosome association analysis shows enhanced ribosome recruitment upon PCBP1 depletion. PCBP1 knockdown increases CFIm25 protein in undifferentiated cells and induces macrophage differentiation markers without stimulation. These findings reveal how alternative polyadenylation controls CFIm25 expression during immune cell differentiation by modulating RNA-binding protein interactions and provide insight into post-transcriptional regulation of RNA processing factors. Impact statement This work reveals how a key regulator of mRNA processing is itself controlled through a previously uncharacterized mechanism during immune cell differentiation. Our findings provide insights into the molecular circuits governing macrophage development and identify potential therapeutic targets for inflammatory disorders where myeloid cell differentiation is dysregulated.
Exposure to various environmental factors and endogenous agents can lead to double-strand DNA breaks. Bacteria are capable of restoring their genome integrity through a process known as the SOS response, which requires t...Exposure to various environmental factors and endogenous agents can lead to double-strand DNA breaks. Bacteria are capable of restoring their genome integrity through a process known as the SOS response, which requires the RecA recombinase. Another protein critical for DNA repair is SMC-like RecN, which facilitates the location of the homologous DNA template by RecA. At present, the function and underlying mechanisms of RecN remain poorly understood. In this work, we use optical tweezers to demonstrate predominant binding of RecN to ssDNA and also show weak binding to dsDNA, resulting in a condition resembling DNA loop formation.
Dysregulation of the transcription factor MYB plays a critical role in leukemia pathogenesis, progression, and prognosis; however, the detailed regulatory mechanisms of MYB remain unclear. Recently, we identified an enha...Dysregulation of the transcription factor MYB plays a critical role in leukemia pathogenesis, progression, and prognosis; however, the detailed regulatory mechanisms of MYB remain unclear. Recently, we identified an enhancer long noncoding RNA (lncRNA) MY34UE-AS, which upregulates MYB expression. Here, we demonstrate that non-POU-domain-containing octamer binding protein (NONO) binds to MY34UE-AS through its RNA recognition motif 2 (RRM2) domain, thereby upregulating MYB expression and splicing. This interaction drives leukemia cell proliferation and migration. Our findings unveil a novel regulatory mechanism of MYB and propose the NONO-MY34UE-AS axis as a potential therapeutic target for leukemia. Impact statement Our study uncovers the NONO-MY34UE-AS-MYB regulatory axis in leukemia, revealing a new layer of MYB control. This mechanistic insight advances understanding of oncogenic transcription factor dysregulation and highlights potential therapeutic targets, offering new directions for leukemia research.
Aging is accompanied by profound changes in both the gut microbiome and the immune system, which engage in continuous, bidirectional communication. Alterations in microbial diversity and metabolism, particularly reductio...Aging is accompanied by profound changes in both the gut microbiome and the immune system, which engage in continuous, bidirectional communication. Alterations in microbial diversity and metabolism, particularly reductions in short-chain fatty acid (SCFA) producers as well as shifts in bile acid and tryptophan-metabolizing species, can incite and worsen inflammation, damage barrier integrity, and accelerate immunosenescence. Concomitantly, immune aging and reduced mucosal IgA promote microbial dysbiosis, forming a self-reinforcing cycle that fuels chronic inflammation ("inflammaging"). Microbial metabolites such as SCFAs, secondary bile acids, and indole derivatives play central roles in this gut-immune dialog, influencing regulatory T-cell balance, epithelial repair, and neurological health through the gut-brain axis. Emerging evidence suggests that diet, probiotics, postbiotics, and microbiome transplantations can restore beneficial microbial and, consequently, immune functions, offering opportunities to promote healthy aging and potentially reverse adverse symptoms. Understanding and targeting the gut microbiome-immune feedback loops may reveal new strategies to modulate inflammaging and extend health span.
Post-translational modifications regulate tau aggregation and propagation, yet how amyloid pathology shapes the tau modification landscape remains unclear. Using liquid chromatography-tandem mass spectrometry, we compare...Post-translational modifications regulate tau aggregation and propagation, yet how amyloid pathology shapes the tau modification landscape remains unclear. Using liquid chromatography-tandem mass spectrometry, we compared tau modifications in MAPT knock-in (MAPT KI) mice and MAPT/App double knock-in mice with App amyloid pathology. Only subtle differences were detected, with a tendency toward increased acetylation within the repeat domain. Because K321 and K331 lie in the fibril-forming core, their roles were further examined. Acetylation at these sites was absent in cynomolgus monkey brains. To test functional relevance, we generated acetylation-mimicking MAPT knock-in mice on the MAPT KI background. Despite tau expression, these mice showed reduced tau phosphorylation at 24 months, with unchanged insoluble tau and seeding activity. Thus, K331 acetylation does not promote tau pathology.
Mono(2-hydroxyethyl) terephthalate (MHET) esterases (MHETases) are enzymes implicated in polyethylene terephthalate (PET) biodegradation. The present study elucidates the structural determinants that result in increased...Mono(2-hydroxyethyl) terephthalate (MHET) esterases (MHETases) are enzymes implicated in polyethylene terephthalate (PET) biodegradation. The present study elucidates the structural determinants that result in increased MHET degradation by a feruloyl esterase, which has been engineered to resemble the MHETase active site. The crystal structures of the variant in apo- and benzoic acid bound states reveal the changes induced by the introduced mutation, specifically the formation of a hydrogen bond and a trans to cis isomerization of a peptide bond in the vicinity of the catalytic site. Molecular dynamics simulations demonstrate the stabilization of the loop harboring the engineered residue, as well as an expansion of the substrate binding cleft, which would facilitate accommodation of a broader variety of substrates, indicative of a promiscuous biocatalyst.
The proteins Inturned and Fuzzy are members of the tri-longin domain (TLD) RabGEF family and activate the GTPase Rab23 downstream of the core planar cell polarity (PCP) proteins Vangl2 and Prickle. To gain insight into t...The proteins Inturned and Fuzzy are members of the tri-longin domain (TLD) RabGEF family and activate the GTPase Rab23 downstream of the core planar cell polarity (PCP) proteins Vangl2 and Prickle. To gain insight into the function of a predicted PDZ domain unique to Inturned among TLD proteins, we performed structural and biochemical characterisations. We show that this domain does not interact with membranes or Vangl2. Instead, we find a phosphorylation-dependent interaction between Vangl2 and a PDZ domain of the apical-basal polarity protein Scribble. A crystal structure of Intu-PDZ reveals a unique PDZ-like fold lacking an interaction site for PDZ-binding motifs. Our data provide new insight into the role of PDZ domains in coordinating cell polarity downstream of Vangl2.