Mitosis is tightly regulated at multiple levels to ensure chromosome stability. The transient phosphorylation of histone H3 at Threonine 3 (H3T3) during cell division is critical for proper chromosome condensation and th...Mitosis is tightly regulated at multiple levels to ensure chromosome stability. The transient phosphorylation of histone H3 at Threonine 3 (H3T3) during cell division is critical for proper chromosome condensation and the accurate segregation of sister chromatids. While Haspin has been identified as the kinase responsible for H3T3 phosphorylation during mitosis, the phosphatases that counteract this modification to maintain balanced phosphorylation levels remain under investigation. In this study, we systematically screened phosphatases encoded in the human genome and identified the nuclear phosphatase SCP4 as an H3T3 phosphatase. SCP4 modulates H3T3 phosphorylation levels and influences the chromosomal recruitment of chromosomal passenger complex (CPC) during mitosis. Aberrant SCP4 expression leads to defective chromosome separation during metaphase and chromosome lagging in anaphase, resulting in aneuploidy. Notably, in SCP4 knockout mice, zygotes exhibit mitotic defects during the first cleavage at the two-cell stage, highlighting SCP4's essential role in ensuring faithful cell division. In summary, we identify SCP4 as a novel phosphatase regulating H3T3 phosphorylation and chromosome dynamics during mitosis, providing new insights into mechanisms safeguarding genomic stability.
Mycobacteria exploit host organelles to survive and proliferate within the intracellular environment. In parallel, host cells rely on these organelles to ensure essential biological functions and mount effective immune d...Mycobacteria exploit host organelles to survive and proliferate within the intracellular environment. In parallel, host cells rely on these organelles to ensure essential biological functions and mount effective immune defenses against invading pathogens. The dynamic competition between host and mycobacteria for control of these organelles represents a central battleground in infection biology and is of increasing scientific interest. Important organelles such as mitochondria, the endoplasmic reticulum, and the Golgi apparatus play pivotal roles in determining the outcome of infection and can tip the balance between pathogen clearance and intracellular survival. Beyond their classical functions in energy production, calcium homeostasis, and protein trafficking, these structures actively participate in immune signaling, metabolism reprogramming, and inflammatory responses. Consequently, they function as powerful defenders against pathogens and, under certain conditions, represent unintentional allies. This review categorizes organelle contributions into two major areas: host-driven cellular defense mechanisms and pathogen-mediated subversion strategies. Recent work in the field is discussed, providing new insights into host-pathogen dynamics and identifying potential therapeutic targets for improved control of mycobacterial infections.
Rapid proliferation of CD8 T cells is crucial for adaptive immunity against viral infection. CD8 T cells can complete division cycles in less than 6 h, representing a physiological extreme for somatic mammalian cells. Em...Rapid proliferation of CD8 T cells is crucial for adaptive immunity against viral infection. CD8 T cells can complete division cycles in less than 6 h, representing a physiological extreme for somatic mammalian cells. Embryonic stem cells utilize specialized cell cycle control mechanisms, including subdued periodic expression, for rapid cell division cycles. CD8 T cell cycle control remains poorly understood. Here, we test whether CD8 T cells utilize embryonic mechanisms to promote rapid cell cycles. We comprehensively measure protein abundances in G1, S, and G2&M phases in three murine cell types: CD8 T cells, embryonic stem cells, and fibroblasts. We discover striking similarities between mESC and CD8 T cells. We demonstrate that CD8 T cells express Cyclin E1 and Emi1/Fbxo5 at high levels to promote S-phase entry. Interestingly, CD8 T cells and mESCs differ in the frequency of G2&M phase cells, the abundance of DNA replication origin licensing and initiation factors, and the abundance of APC/C substrates. Thus, somatic T cells have both unique and shared cell cycle control mechanisms to promote rapid cell cycles.
The evolution of allele frequencies in a population is often ascribed to differential fitness among organisms carrying different alleles. However, selection during meiosis can substantially influence this process. Here,...The evolution of allele frequencies in a population is often ascribed to differential fitness among organisms carrying different alleles. However, selection during meiosis can substantially influence this process. Here, we studied the evolution of a hybrid yeast population over six meiotic generations and observed rapid allele frequency dynamics at many genomic loci. By tracking the whole population and analyzing single gametes we discovered that biased segregation pattern during meiosis can drive rapid evolutionary changes at translocation-linked loci. The inter-chromosomal translocation present in one parental strain creates quadrivalent structures that promote adjacent-1 segregation coupled with crossover formation over alternate segregation during meiosis. This ultimately increases the proportion of unbalanced gametes, and consequently alters allele frequencies in the population. These findings demonstrate that meiotic selection operates more broadly than previously recognized and constitutes a significant evolutionary force affecting population allele frequencies. Given the prevalence of inter-chromosomal translocations, biased segregation pattern may complement the established role of translocations in shaping evolutionary outcomes.
Jin X, Han P, Wang Y
… +16 more, Wang H, Zhang C, Di Maio A, Yu J, Hao T, Gu Y, Zhang Z, Zhang W, Qi J, Bi Y, Zhang X, Sun L, Wang N, Liu Y, Song H, Gao GF
Clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) continue to circulate worldwide, posing zoonotic threats, especially with recent cattle outbreaks. The mechanisms by which these viruses adapt to mam...Clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) continue to circulate worldwide, posing zoonotic threats, especially with recent cattle outbreaks. The mechanisms by which these viruses adapt to mammalian hosts while maintaining a broad avian tropism remain poorly understood. Here, we demonstrate that two naturally occurring mutations (K222Q and S227R) in the hemagglutinin (HA) of a human-infecting H5N8 strain, first identified in 2020, enhance binding affinity for both α2-6-linked and Sialyl Lewis (SLe) glycans, which may underlie the broad tissue binding and cross-species potential. Structural analyses reveal that these mutations expand receptor specificity for these glycans, which are abundant in the human respiratory tract and duck trachea, providing a possible molecular basis for cross-species transmission. Our findings suggest that clade 2.3.4.4b H5Ny viruses evolved dual receptor specificity as early as the 2020 Russian H5N8 strain, potentially contributing to sporadic human infections and widespread dissemination among birds and mammals.
Fleetwood AJ, Mukhamedova N, Dragoljevic D
… +12 more, Lee MKS, Xu Y, Dona MSI, Hsu I, Bertuzzo Veiga C, Takeuchi F, Crossett B, Tran D, Pinto AR, Bukrinsky M, Murphy AJ, Sviridov D
HIV infection is accompanied by chronic inflammation-related co-morbidities, even when viral replication is suppressed by therapy. This persistent inflammatory state suggests that long-lived immune cell lineages may acqu...HIV infection is accompanied by chronic inflammation-related co-morbidities, even when viral replication is suppressed by therapy. This persistent inflammatory state suggests that long-lived immune cell lineages may acquire stable pro-inflammatory programming. Here, we investigate whether inflammatory programming can be imprinted within hematopoietic lineages, following the exposure of mice and bone marrow-derived macrophages (BMDMs) to extracellular vesicles (EVs) carrying Nef, a key inflammatory factor of HIV. Multi-omics profiling shows that hematopoietic cells exposed to Nef-EVs undergo epigenetic remodeling and reprogramming of energy and lipid metabolism characteristic of trained innate immunity. The inflammatory phenotype in BMDMs is partially reversed by inhibition of glycolysis, a key metabolic driver of trained immunity. We demonstrate that following competitive bone marrow transplantation, hematopoiesis in mice receiving bone marrow from Nef-EV-treated donors displays a sustained bias toward myelopoiesis, and BMDMs retain enhanced inflammatory potential. These findings demonstrate that Nef-EVs can imprint a lasting inflammatory memory, mechanistically similar to trained immunity, in hematopoietic cells. This memory persists beyond the initial exposure and may contribute to chronic inflammation in people with HIV.
Stress granules (SGs) are transient cytoplasmic biomolecular condensates that play a role in the cellular response to proteotoxic stress. It has been previously shown that ubiquitination regulates SG dynamics; however, t...Stress granules (SGs) are transient cytoplasmic biomolecular condensates that play a role in the cellular response to proteotoxic stress. It has been previously shown that ubiquitination regulates SG dynamics; however, the specific mechanisms by which ubiquitin affects SGs are not fully understood. Here, using proximity proteomics, we discover that the recruitment of several E3 ubiquitin ligases, VCP cofactors and HSP70 to SGs is dependent on the activity of the E1 ubiquitin ligase UBA1. The RNA-binding E3 ubiquitin-protein ligase Makorin 2 (MKRN2) is strongly depleted from SGs in the absence of UBA1 function. MKRN2 promotes both the proper formation of SGs and their disassembly following stress recovery, by preventing the accumulation of misfolding-prone defective ribosomal products (DRiPs) within SGs. Therefore, MKRN2 is a novel regulator of SGs that mediates the maintenance of granulostasis.
The chemical composition of membrane lipids differs between eukarya, bacteria and archaea. The central dogma posits that the stereochemistry of phospholipids in bacteria is distinct from archaea. Bacterial phospholipids...The chemical composition of membrane lipids differs between eukarya, bacteria and archaea. The central dogma posits that the stereochemistry of phospholipids in bacteria is distinct from archaea. Bacterial phospholipids consist of fatty acid lipid tails esterified to the sn-glycerol 3-phosphate lipid backbone (G3P), whereas archaeal phospholipids comprise isoprenoid lipid tails ether-linked to the stereochemical different sn-glycerol 1-phosphate (G1P). This segregation, the "lipid divide", is however not as strict as previously thought. Recent reports demonstrate that both glycerol-phosphate backbones are present in phospholipids from various Gram-positive bacteria. To test if the stereochemical variability can be attributed to conventional lipid biosynthesis, we characterize the stereospecificity of the relevant glycerol-phosphate acyltransferases PlsY and PlsB, as well as the lysophosphatidic acid acyltransferase PlsC, catalyzing the key steps in phospholipid biosynthesis yielding phosphatidic acid, both in the Gram-positive B. subtilis and the Gram-negative E. coli. While PlsB is strictly stereospecific for glycerol 3-phosphate, PlsY and PlsC can utilize both stereo-forms of the glycerol-phosphate. Hence, the variability in lipid backbone stereochemistry is an intrinsic part of bacterial phospholipid biogenesis, questioning the supposedly strict stereochemical segregation of bacteria and archaea after the lipid divide.
Patients with lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency typically suffer from severe B cell dysfunction. However, the underlying mechanisms remain incompletely understood. In this study, w...Patients with lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency typically suffer from severe B cell dysfunction. However, the underlying mechanisms remain incompletely understood. In this study, we identify non-muscle myosin IIA (NMIIA) as an interaction partner of LRBA in B cells, and uncover a role for LRBA in regulating actin cytoskeleton dynamics during B cell activation. LRBA-deficient B cells exhibit abnormal migration, impaired F-actin polymerization, and reduced B cell receptor signalling and polarization upon activation. In addition, LRBA deficiency severely disrupts immune synapse formation as evidenced by diminished central SMAC formation, reduced microtubule organizing center translocation and disrupted BCR and lysosome polarization. Consistent with these defects, internalization of the BCR-antigen complex is also impaired. Mechanistically, NMIIA activation, assessed by myosin light chain (MLC) phosphorylation, is reduced in LRBA-deficient cells. In addition, LRBA co-localizes with active NMIIA during both migration and immune synapse formation. Collectively, our findings establish LRBA as an important regulator of cytoskeleton dynamics during B cell activation, which may contribute to the defective humoral immunity observed in LRBA-deficient patients.
Kolb S, Diekmann L, Lochert EE
… +12 more, Warmuth L, Ritter J, Schmidtke G, Weber M, Hoffmann M, List M, Kotlarz D, Serr I, Daniel C, Busch DH, Schmidl C, Schumann K
The chromatin organizer SATB1 is indispensable for thymic regulatory T cell (Treg cell) development and T helper cell induction. Several gene loci have been described to be SATB1-controlled, including the transcription f...The chromatin organizer SATB1 is indispensable for thymic regulatory T cell (Treg cell) development and T helper cell induction. Several gene loci have been described to be SATB1-controlled, including the transcription factor GATA3 and the cytokine loci IL-4 and IL-17. However, the global effects of SATB1 on fully differentiated human CD4 conventional T cells (Tconv cells) and Treg cells, and thus the potential of SATB1 as a target for T-cell engineering, are poorly understood. Here, we describe SATB1-regulated gene signatures as largely subset-specific, with broader effects on Treg cells. Despite distinct gene-regulatory patterns, we observe overarching dysregulated cytokine and JAK-STAT signaling after SATB1 ablation. Functionally, SATB1 KO reduces suppressive capacities of human Treg cells but boosts tumor clearance via CD4 CAR T cells in a preclinical, humanized mouse model. Taken together, Treg destabilization and simultaneous increased activation of CD4 CAR T cells by SATB1 modulation may be a strategy to boost the efficiency of CAR T cell therapies.
The evolution of new enzymatic functions is constrained and guided by the architecture of an organism's metabolic and regulatory networks and environmental constraints. Here, we identify a kinase that has evolved from py...The evolution of new enzymatic functions is constrained and guided by the architecture of an organism's metabolic and regulatory networks and environmental constraints. Here, we identify a kinase that has evolved from pyruvate phosphate dikinase. Using biochemical and systems-level analyses, we show that this enzyme, encoded by rv1127c in Mycobacterium tuberculosis (Mtb), has diverged from its ancestral role in central carbon metabolism to function as a histidine kinase in pathogenic mycobacteria and related species. We designate this enzyme Virulence Associated DiKinase (VadK), reflecting its ability to autophosphorylate and its role in virulence. VadK is essential for the utilization of carbon sources critical for survival within the host and to cause tuberculosis (TB) in murine models. Furthermore, VadK interacts with enzymes of the methylcitrate cycle, and C-tracer experiments demonstrates that it fine-tunes flux through this pathway, with elevated flux proving growth limiting. Together, these findings identify VadK as a regulatory kinase that integrates metabolic control with virulence in Mtb, revealing a new facet of metabolic regulation in bacterial pathogenesis and a potential target for therapeutic intervention.
Predicting the fitness effects of mutations is central to understanding molecular evolution and interpreting genome sequence data. Such predictions remain challenging due to the inter-dependent roles of coding and non-co...Predicting the fitness effects of mutations is central to understanding molecular evolution and interpreting genome sequence data. Such predictions remain challenging due to the inter-dependent roles of coding and non-coding genetic variation. While coding mutations alter protein structure, stability, activity, and sometimes abundance, regulatory mutations modulate gene expression timing and levels. Because coding and regulatory variation are thought to independently impact features of protein function, their combined effects or complex phenotypes are often unexpected. In particular, regulatory-coding epistasis, whereby the fitness effect of a coding mutation depends on the regulatory background, can reshape fitness landscapes and influence adaptive trajectories. In this review, we explore how variation in protein abundance and activity jointly shape fitness, constrain adaptation, and impact molecular evolution. Drawing on examples from systematic studies carried out in unicellular organisms, we speculate on a fitness function integrating abundance and activity and discuss the broader implications of these interactions for evolutionary dynamics, genetic disease, and phenotypic diversity.
Systemic RNA movement offers a route for non-transgenic trait improvement by grafting, but progress has been limited by the lack of reliable tools to identify mobile RNA elements. Here, we report a fluorescence-aptamer-b...Systemic RNA movement offers a route for non-transgenic trait improvement by grafting, but progress has been limited by the lack of reliable tools to identify mobile RNA elements. Here, we report a fluorescence-aptamer-based live-imaging platform for screening mobile RNAs and mapping mobility elements. In a transient screening assay, live imaging of 100 RNA-seq-predicted candidates detects three reproducibly mobile mRNAs, CAT3, CK1, and GAI, under our assay conditions, with GAI mRNA exhibiting the highest bidirectional mobility. Truncation analysis defines two independent 30-nt cis-elements, GME1 in the coding region and GME2 in the 3'UTR, with GME2 mediating a higher transport rate and mobility rate. A tandem 2×GME2 cassette in the 3'UTR functions as a high-capacity RNA delivery module and, in our transient assay, outperforms the tRNA-like sequence motif (TLS; tRNA) construct tested here. Using 2×GME2, we deliver three otherwise non-mobile mRNAs across graft junctions and detect associated molecular or phenotypic effects in recipient tissues. Together, these findings establish a live-imaging-guided pipeline for mobile RNA validation and identify 2×GME2 as a non-viral RNA delivery element for graft-mediated trait modification.
Metabolic reprogramming, including enhanced glycolysis and altered fatty acid metabolism, supports the proliferation of cancer cells under hypoxic stress. However, the mechanism underlying the regulation of cholesterol m...Metabolic reprogramming, including enhanced glycolysis and altered fatty acid metabolism, supports the proliferation of cancer cells under hypoxic stress. However, the mechanism underlying the regulation of cholesterol metabolism under hypoxic stress remains incompletely understood. Here, we report that lactate-induced cholesterol accumulation activates mammalian target of rapamycin complex 1 (mTORC1) signalling under hypoxic conditions, thereby promoting hepatocellular carcinoma (HCC) progression. Mechanistically, lactate upregulates scavenger receptor class B type 1 (SCARB1) expression by increasing histone H3 lysine 18 lactylation (H3K18la), leading to increased cholesterol levels. We further demonstrate that SCARB1-mediated cholesterol uptake is essential for the activation of mTORC1, which promotes tumour growth by preventing excessive autophagy in HCC cells. Importantly, analysis of clinical HCC samples reveals a positive correlation between H3K18la expression and SCARB1 expression. Taken together, these findings provide novel insights into hypoxia-driven metabolic reprogramming and reveal a previously unrecognized connection between lactate and cholesterol metabolism, suggesting a potential innovative cancer therapy for HCC.
The MINDY family of deubiquitinases (DUBs) are exemplified by their preference for cleaving K48-linked polyubiquitin. MINDY3 is architecturally distinct from other MINDY DUBs as its catalytic domain spans the entire leng...The MINDY family of deubiquitinases (DUBs) are exemplified by their preference for cleaving K48-linked polyubiquitin. MINDY3 is architecturally distinct from other MINDY DUBs as its catalytic domain spans the entire length of the protein except for an atypical EF-hand insertion. We uncover this EF-hand (MINDY3) to be a ubiquitin-binding domain with three distinct binding sites, enabling MINDY3 to bind and effectively cleave long polyubiquitin chains. Furthermore, the MINDY3 domain binds not only to polyubiquitin but also to the UBL domain of the proteasome shuttling and DNA repair factors RAD23A and RAD23B. The MINDY3 facilitates this interaction with RAD23s in cells and mediates MINDY3 recruitment to DNA damage sites, establishing this unique DUB as a potential regulator of cellular DNA damage responses. MINDY3 binds specifically to the UBL domain of RAD23s, and none of the other UBLs tested. The crystal structure of the MINDY3:RAD23A domain complex reveals the molecular basis for specificity. We find that MINDY3 can form a ternary complex with RAD23A/B and polyubiquitin, and our findings suggest a model wherein MINDY3 can deubiquitylate RAD23A/B-bound clients.
Cytotoxic CD8 T lymphocytes (CTLs) efficiently eliminate infected and cancerous cells throughout the body. T cell receptor (TCR)-induced Hedgehog signalling contributes to CTL-mediated killing, but how the pathway is act...Cytotoxic CD8 T lymphocytes (CTLs) efficiently eliminate infected and cancerous cells throughout the body. T cell receptor (TCR)-induced Hedgehog signalling contributes to CTL-mediated killing, but how the pathway is activated downstream of the TCR is unknown. Here, we show that extracellular calcium (Ca) flux through L-type voltage-gated Ca (Ca1) channels at the plasma membrane downstream of the TCR drives induction of the Hedgehog transcription factor Gli1, which is important for CTL killing in vitro and in vivo. This previously unknown non-canonical Hedgehog pathway is independent of canonical signalling and represents a primary mechanism of Gli1 induction in naive CD8 T cells, whereas CTLs can also activate Gli1 via MAPK. We further show that Ca1 channel-controlled Gli1 induction is functionally important for CTL killing in mice and humans and other cytotoxic lymphocytes. Notably, killing capacity can be amplified using a small molecule Ca1 agonist or by overexpressing a gain-of-function Ca1 subunit. These findings suggest a strategy to improve cytotoxic lymphocyte function in the clinic, including in CAR T cell therapy.
Lentini G, Coppolino F, Famà A
… +11 more, De Gaetano GV, Grasso F, Fiore L, Berbiglia A, Carnevale S, Venza I, Lien E, Venza M, Jaillon S, Teti G, Beninati C
Caspase-8 is essential for maintaining organismal integrity by preventing cell death and subsequent inflammation in specific epithelial and endothelial tissues. Here, we show that caspase-8 also controls a systemic, cell...Caspase-8 is essential for maintaining organismal integrity by preventing cell death and subsequent inflammation in specific epithelial and endothelial tissues. Here, we show that caspase-8 also controls a systemic, cell death-independent inflammatory pathway that is constitutively active during homeostasis. In vivo, selective caspase-8 inhibition produces, in the absence of other stimuli, marked neutrophilia driven by circulating proinflammatory and chemotactic cytokines and promotes bacterial clearance during infection. In vitro, caspase-8 inhibition triggers in neutrophils, but not in macrophages, a profound transcriptional response associated with the release of IL-1β and other cytokines. This process requires tonic TNF-α production by neutrophils, which acts autocrinally to sequentially activate RIPK1, RIPK3, MAPKs, and NF-κB. The IL-1β release induced by caspase-8 inhibition requires gasdermin D and neutrophil serine proteases, but not canonical inflammasome components. Our data uncover the mechanistic features of a neutrophil-centric, proinflammatory pathway that can be therapeutically targeted to augment host defenses against pathogens.
Intracellular pathogens such as Legionella pneumophila secrete effector proteins that manipulate host cell processes to promote bacterial survival. One such effector, RidL, is known to inhibit retrograde trafficking by i...Intracellular pathogens such as Legionella pneumophila secrete effector proteins that manipulate host cell processes to promote bacterial survival. One such effector, RidL, is known to inhibit retrograde trafficking by interacting with the retromer complex via its N-terminal domain. Here, we identify a second function of RidL mediated by its C-terminal domain, which directly binds to the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) and related large GTPases. In vitro, RidL reduces Drp1 GTPase activity and disrupts its oligomerization. During infection, RidL localizes to mitochondria, enhances the accumulation of Drp1 and the outer membrane protein Tom20, and impairs mitochondrial dynamics and function. Moreover, in L. pneumophila-infected cells, RidL promotes phosphorylation of Drp1 at Ser616, leading to Drp1 activation and mitochondrial fragmentation. These findings establish RidL as a bifunctional effector that targets both the retromer complex and Drp1 through distinct domains. By interfering with host mitochondrial dynamics, RidL enables L. pneumophila to remodel host organelles and optimize conditions for intracellular replication.
Chromosomal instability (CIN), characterized by frequent changes in chromosome number and structure, is common in human carcinomas and often leads to aneuploidy, an unbalanced number of chromosomes. Drosophila has been i...Chromosomal instability (CIN), characterized by frequent changes in chromosome number and structure, is common in human carcinomas and often leads to aneuploidy, an unbalanced number of chromosomes. Drosophila has been instrumental in demonstrating that CIN can promote tumour growth and malignancy through aneuploidy-induced senescence, a state marked by cell-cycle arrest and high secretory activity. Despite extensive chromosomal heterogeneity, we show that these cells share a distinct transcriptional programme, with most responses to aneuploidy and senescence regulated at the transcriptional level. We unravel a pro-survival function of the Hippo-Yorkie signalling pathway in aneuploidy-induced senescent cells and present evidence that nearly 10% of the most upregulated genes encode secreted proteins of the senescence-associated secretory phenotype. Five of these proteins act additively, locally or systemically, to block proliferation and induce cell death in neighbouring tissues. This non-autonomous cell death feeds back to the tumour to enhance its growth, resembling super-competition and providing insight into tumour-host interactions relevant to human cancer.