Ribonucleotide reductases (RNRs) convert all four ribonucleotides to deoxyribonucleotides, providing essential building blocks for DNA biosynthesis and repair through radical-based catalysis. These functions are key to cellular proliferation and have made RNRs well established targets for antimicrobial and antiviral drugs and combination chemotherapies. Here, we describe a novel highly sensitive one-pot enzymatic assay, which amplifies RNR activity by coupling it to the synthesis of a fluorogenic RNA aptamer. We validated this approach by testing RNR activity under dNTP-limiting conditions to emulate RNR's complex allosteric regulatory patterns and by detecting the dose- and time-dependent inhibition of RNR by hydroxyurea. This unique assay builds on previous high-throughput screening assays for investigation of RNR's catalytic mechanisms by improving sensitivity and reducing readout timeframes. Impact statement Ribonucleotide reductases (RNRs) are essential for controlling cellular dNTP supply and are major targets in cancer, antiviral, and antimicrobial therapy. FLARE is a novel single-tube, real-time RNR assay, coupling dNTP synthesis to the transcription of a fluorogenic aptamer for continuous monitoring of activity, regulation, and inhibition using standard microplate readers.

The long lifespan of humans is often not matched with health span. Thus, there is a need for rejuvenation strategies. Here, we first discuss the evolutionary benefits of the long human lifespan, particularly when coupled with an extended health span. We then highlight the importance of understanding the complexity of aging before interfering with it. This raises the question of the optimal target for rejuvenation. We propose the blood system and hematopoietic stem cells (HSCs). Their decline is associated with dysfunction and disease in other organs, crystallizing them as a central player in organismal aging. We present rejuvenation strategies targeting the hematopoietic system, especially HSCs, and explore their systemic benefits. Overall, we summarize the potential of the blood system to reverse aging. Impact statement There is a current need to reduce the economic burden caused by aging-related diseases. In this perspective article, we discuss the evidence that supports that rejuvenating or delaying aging of the blood system has a beneficial and systemic impact on human health.

Protein pyrophosphorylation is an emerging, unusual posttranslational modification. This signaling mechanism can be driven by inositol pyrophosphate messengers, which can convert a prephosphorylated protein to the corresponding pyrophosphoprotein. Endogenous protein pyrophosphorylation influences various cellular processes and signaling pathways, including the regulation of rRNA synthesis and the modulation of vesicular trafficking. Herein, we will summarize the current detection and analysis methods that have established the occurrence of pyrophosphorylation. These methods have also been used to explore the effects of pyrophosphorylation on protein structure and function. Putative mechanisms for the regulation of this intriguing, understudied modification will be discussed. Finally, the future needs for this developing area of signal transduction research are highlighted.

HipA-like kinases are widespread bacterial serine-threonine kinases, yet their regulatory mechanisms remain poorly understood. Here, we characterise two novel HipA-like systems, the monocistronic hipL and bicistronic hipIN, also encoding HipS-like and HIRAN domains. We show that the hipL gene contains an internal translation initiation site producing a smaller variant, HipL, which counteracts HipL-mediated toxicity via its HipS-like domain. Contrary to this, HipN requires both the HipS-like and the HIRAN domains to neutralise HipI-mediated toxicity. Neither system forms stable toxin-antitoxin (TA) complexes in vitro, distinguishing them from classical type II systems. Finally, we show that autophosphorylation affects HipL but not HipI-mediated toxicity. These findings reveal diverse regulatory architectures in HipA-like TA systems, shaped by domain composition and operon structure. Impact statement Kinases are increasingly recognised as key regulators in bacteria. Here, we show how complex operon and domain structures can contribute to kinase function and regulation, revealing increasingly complex regulatory networks in microbes.

Intrinsically disordered protein regions (IDRs) are found across all domains of life and are characterized by a lack of stable 3D structure. Nevertheless, IDRs play critical roles in the most tightly regulated cellular processes, including in the core circadian clock. The molecular oscillator at the heart of circadian regulation leverages IDRs as dynamic interaction modules-for activation and repression, alike-to support robust timekeeping and expand clock output and regulation. Here, we cover the biophysical mechanisms conferred by IDRs and their modulators. We survey the IDRs in clock proteins that are widely prevalent from fungi to mammals and discuss the importance of IDRs to the core clock and beyond.

The ability to align circadian phase to specific cues, or 'entrainment', is a defining feature of a circadian rhythm. Entrainment is critical for useful circadian function, as it enables organisms to determine the specific time of day to perform temporally restricted behavioural and physiological activities, ranging from sleep to cell division. While mammals have long been known to entrain their circadian rhythm, recent work has shed light on how this is achieved in every single cell, all of which maintain their own individual circadian oscillation. Here I will highlight the current understanding of how the major entraining cues of light, feeding and temperature are communicated to cells to alter their phase. Knowledge of the mechanisms of cellular entrainment has the capacity to impact both fundamental understanding of circadian rhythms and our application of cellular circadian research to real-world problems, including shift work.

The emergence of land plants from aquatic habitat over half a billion years ago marked a pivotal moment in Earth's history, profoundly altering both soil composition and atmospheric chemistry. This remarkable evolutionary transition was accompanied by the establishment of multiple signaling pathways that facilitated plant adaptation to terrestrial environments. Among these, signaling pathways based on myo-inositol-derived cellular messengers have recently gained significant attention. Combinatorial attachment of phosphate to the myo-inositol (hereafter inositol) ring produces a large array of inositol phosphate (InsP) messengers that are involved in an ever-growing number of physiological processes in eukaryotes. Whether these messengers contributed to the emergence of land plants remains an open question. In this review, we explore recent advances in understanding the astonishing molecular diversity, biosynthesis and regulation of InsP molecules in plant cells, their integration into various physiological pathways, and the potential implications of InsP signaling pathways in the evolution of land plants.

High glucose-induced endothelial dysfunction is a hallmark of diabetic microvascular complications. Reduced hydrogen sulphide (HS) levels and dysregulation of the HS-producing enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) are linked to this dysfunction. Dihydrolipoic acid (DHLA), formed from alpha-lipoic acid in mitochondria, has been proposed to enhance HS release from 3-MST; however, this has not been empirically tested in diabetes. Using a cell culture model, this study demonstrates that DHLA supplementation enhances HS production via 3-MST and improves endothelial function under high glucose conditions. Our results provide foundational knowledge and indicate that targeting the 3-MST/HS pathway via DHLA supplementation may have therapeutic potential to reduce or slow the onset and progression of diabetic microvascular complications.

Vesicle fusion events are crucial for the survival of Giardia lamblia as they drive nutrient uptake and morphological stage transitions. Unlike most eukaryotes, Giardia has a minimal vesicular trafficking machinery. We report a rare exception to this minimalism wherein two paralogues of N-ethylmaleimide-sensitive factor (NSF) are present in this parasite. Localization studies indicate that these highly homologous paralogues-GlNSF and GlNSF-likely function independently under various stress conditions, as GlNSF remains at peripheral vesicles, while the major pool of GlNSF redistributes to anterior flagella-associated structures. These paralogues also exhibit selective affinity for the α-soluble NSF attachment proteins (Glα-SNAPs). This selectivity stems from sequence divergences near their N termini. The two GlNSFs colocalize and coimmunoprecipitate, indicating the presence of a heterohexameric 20S complex in trophozoites. This study is the first to report the presence of a heterohexameric 20S complex and reveals adaptive specialization of vesicle trafficking machinery within a reduced eukaryotic system. Impact statement Here we report that a unicellular parasitic protist, Giardia lamblia, has two NSF paralogues, which is a rarity in eukaryotes. Although they share a high degree of homology, they are likely to discharge independent functions, especially under stress conditions.

It has been proposed that the regulatory Sit4-associated protein subunit 3 (SAPS3) of protein phosphatase 6 (PP6C) acts as an AMP-activated protein kinase (AMPK) inhibitor by recruiting PP6C to dephosphorylate AMPKα-T172. While we confirm this interaction in HEK293 cells, we find limited evidence for a SAPS3-AMPK interaction in metabolically perturbed liver and skeletal muscle from humans and mice. Across fasting, high-fat diet feeding and exercise conditions, co-immunoprecipitation assays failed to detect endogenous SAPS3-AMPK and PP6C-AMPK interactions. These findings challenge the physiological relevance of SAPS3/PP6C as regulators of AMPK in mature tissues and highlight the need for further investigation into the regulation of AMPK by protein phosphatases in vivo.

Circadian transcription factors (TFs) orchestrate daily rhythms in gene expression to drive rhythmic biological functions. In mammals, this system relies on the TF CLOCK:BMAL1, which binds E-boxes to initiate rhythmic transcription. While traditionally viewed as a master activator, CLOCK:BMAL1 is now recognized to engage in additional regulatory functions that are essential for its activity. This perspective focuses on the mammalian circadian clock and integrates genomic, structural, and single-molecule footprinting data to highlight emerging insights into how CLOCK:BMAL1 regulates chromatin architecture, cooperates with other TFs, and coordinates complex enhancer dynamics. We propose an updated framework for how circadian TFs operate within dynamic and multifactorial chromatin landscapes, and prime cis-regulatory elements for rhythmic transcriptional bursts. We also discuss how this framework underlies circadian reprogramming and transcriptional plasticity.

The LH2 complex is essential for light harvesting in many photosynthetic bacteria. To elucidate the specific structural role of carotenoids, we analyzed LH2 complexes from Ectothiorhodospira haloalkaliphila with inhibited carotenoid biosynthesis. This approach allowed us to study complexes incorporating the colorless carotenoid phytoene instead of the native, colored pigments. A 1.92 Å cryo-EM reconstruction revealed that phytoene fully substitutes for the native carotenoids while maintaining the octameric symmetry of the complex and the precise arrangement of bacteriochlorophylls. These results demonstrate that the architectural function of carotenoids in LH2 complexes is maintained even when their light-absorption capability is altered, providing new mechanistic insight into the structural basis of pigment-protein interactions in photosynthetic antenna complexes.

Cells are constantly exposed to various sources of DNA damage, including radiation, chemicals, replicative stress and oxidative stress, that threaten genome stability. To ensure faithful DNA repair, transcription regulation needs to be tightly controlled. This regulation involves transcriptional suppression, selective activation of DNA repair-related genes and transcriptional recovery post-repair. Failure to properly modulate transcription during DNA damage can result in collisions between transcriptional and repair machineries, misregulation of repair genes and delayed recovery, ultimately compromising genomic integrity. Chromatin modifications play a central role in this process. These modifications include phosphorylation, methylation, acetylation and ubiquitination, which orchestrate DNA accessibility for repair machinery and fine-tune transcriptional responses. Absence of these modifications leads to inefficient DNA repair and transcriptional errors that are implicated in diseases such as cancer, premature ageing and neurodegenerative disorders. In this review, we delve into the role of various types of histone modifications, such as phosphorylation, methylation, acetylation and ubiquitination and how they regulate transcription in response to DNA damage. Impact Statement This review elucidates how histone modifications orchestrate transcription regulation during DNA damage response, safeguarding genome stability. We also discuss transcription dysregulation in diseases such as cancer and premature aging. Our review provide insights on chromatin-based repair pathways and guide researchers in developing therapeutic targets.

Vibrio cholerae, the causative agent of cholera, uses surface proteins such as the repeats-in-toxin (RTX) adhesin FrhA to colonize hosts and initiate infection. Blocking bacterial adhesion represents a promising therapeutic strategy to treat infections without promoting drug resistance. FrhA contains a peptide-binding domain (PBD) that is key for hemagglutination, human epithelial cell binding, and V. cholerae biofilm formation. Previous studies identified a lead pentapeptide ligand with the sequence Ala-Gly-Tyr-Thr-Asp (AGYTD) that blocks V. cholerae colonization of the mouse small intestine at high micromolar concentrations. In this study, a structure-guided approach identified a minimal D-amino acid-containing tripeptide motif with higher affinity for the FrhA-PBD and predicted metabolic stability. Our results contribute to the development of anti-adhesion strategies to combat infections. Impact statement Our study elucidates the molecular basis of peptide recognition by the Vibrio cholerae adhesin FrhA and develops minimal D-amino-acid peptides that block adhesion with nanomolar affinity. These findings advance understanding of RTX adhesins and provide a structural blueprint for next-generation anti-adhesion therapeutics against cholera and related infections.

Circadian clocks allow for the physiological anticipation of daily environmental changes. A circadian rhythm in intracellular Mg was recently discovered in multiple eukaryotes. Given the pivotal role for Mg in metabolism, Mg rhythms could affect cellular energy expenditure over the daily cycle. To probe the potential mechanisms underlying the generation of cellular Mg rhythms, we present a phylogenetic analysis of Mg transport proteins. Extensive conservation was observed for ancestral prokaryotic proteins, identifying these as candidate proteins mediating Mg rhythms across eukaryotes. We also posit that shared allosteric regulation of Mg transport proteins might underlie Mg rhythms and propose a reciprocal feedback model between the rhythmic usage of Mg and rhythmic transport activity.

F. Zhang , H. Ni , X. Li , H. Liu , T. Xi and L. Zheng , "LncRNA FENDRR Attenuates Adriamycin Resistance via Suppressing MDR1 Expression Through Sponging HuR and miR-184 in Chronic Myelogenous Leukaemia Cells," FEBS Letters 593, no. 15 (2019): 1993-2007, https://doi.org/10.1002/1873-3468.13480. The above article, published online on 10 June 2019 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, T. Xi and L. Zheng; the journal Editor-in-Chief, Michael Brunner; the Federation of European Biochemical Societies; and John Wiley & Sons Ltd. The retraction has been agreed upon following an investigation into concerns raised by a third party. Several duplications were identified between Figures 1B, 2C, 2D, 2G, 3A, 3B, and 7D of this article and figures published in another article by two of the same authors. The authors contacted the journal and explained this was an inadvertent error that occurred during figure preparation as the studies were conducted simultaneously. However, due to the nature and extent of the duplications, the editors no longer have confidence in the results and conclusions reported in the paper. The co-authors, F. Zhang, H. Ni, X. Li and H. Liu did not respond to our notice of retraction.

Mammalian cells express seven distinct phosphoinositide species: PI(3)P, PI(4)P, PI(5)P, PI(3,4)P, PI(3,5)P, PI(4,5)P, and PI(3,4,5)P. With the rapid development of labeling, imaging, and manipulation tools, our understanding of the spatial distribution, functions, and regulation of these phosphoinositides has advanced significantly. Tightly regulated by lipid kinases, phosphatases, and lipid transfer proteins, each phosphoinositide exhibits a unique yet dynamic spatial distribution at both subcellular and suborganelle levels. This distinct spatial organization is critical for controlling membrane trafficking, defining organelle identity and function, mediating signal transduction, and supporting other essential cellular processes. Dysregulation of spatial phosphoinositide signaling has been linked to various human diseases. In this review, we provide a brief overview of current insights into the spatial organization of phosphoinositide signaling, highlighting its key roles in regulating membrane dynamics and signal transduction at the plasma membrane, endosomes and lysosomes, the Golgi apparatus, the ER, and the nucleus.

Photosystem II (PSII) water oxidation includes proton and electron transfer pathways, occurring during the sequential S-state transitions of MnCaO. Here, we investigate the charge separation events during the S to S transition that take place via the STyrZ intermediate, utilizing electron paramagnetic resonance (EPR) spectroscopy. The increasing number of cycles of STyrZ formation and decay results in a gradual diminution of the STyrZ signal intensity which is proportional to the amount of S state. Our results point to the progressive accumulation of a different configuration of the donor side of PSII at the S state that allows the MnCaO to be oxidized. These results consolidate previous investigations supporting that, during the lifetime of the STyrZ, a proton from MnCaO is removed, prior to the advancement to the S state.

Dipeptidyl peptidase IV (DPPIV) family proteases are classically defined by their strict removal of N-terminal dipeptides from substrates bearing a proline or alanine at the P position. Here, we report that both Caenorhabditis elegans DPF-3 and human DPP4 (hDPP4) possess previously unrecognized tripeptidyl peptidase activity in addition to dipeptidyl peptidase activity. This activity plays a key role in the processing of the WAGO-1 protein N-terminus, which is essential for proper small-RNA loading, germline genome defense, and fertility. Kinetic analyses using the fluorogenic substrate H-Met-Gly-Pro-AMC further demonstrated that, in vitro, DPF-3 and hDPP4 can liberate AMC. These findings potentially expand the substrate repertoire of DPPIV proteases, suggesting that these proteases could function as versatile N-terminal processors, with important implications for nascent protein maturation.

It was previously shown that a fructose-rich diet (FRD) induces prediabetes, cardiac dysfunction, and hypertrophy (CH) in males. We assessed FRD and estrogen depletion on female metabolism and cardiovascular function. Females on FRD or control diet (CD) did not develop prediabetes or cardiac dysfunction, although FRD-fed mice showed CH vs. CD. One month of ovariectomy (OVX) did not induce prediabetes, but FRD impaired glucose tolerance in OVX mice without additional metabolic or cardiac changes. Calcium transient amplitude decreased in OVX-FRD vs. SHAM-FRD, with delayed decay, suggesting reduced activity of the sarcoplasmic/endoplasmic reticulum Ca ATPase (SERCA2a). Sodium-hydrogen exchanger 1 (NHE1) expression also decreased in OVX-FRD. These findings indicate estrogen loss does not cause dysfunction but modifies glycemic response to FRD, while reduced NHE1 may help preserve cardiac function. Impact statement In ovariectomized (OVX) mice, estrogen deficiency leads to insulin resistance and impaired glucose tolerance only when combined with a fructose-rich diet (FRD); neither OVX nor FRD alone is sufficient to induce these alterations. However, despite hormonal changes, OVX mice fed a FRD do not develop significant cardiac dysfunction.