
Lipids act as direct receptor ligands that modulate the activity of mammalian membrane-bound adenylyl cyclases, establishing a new level of regulation for cAMP biosynthesis.


coremsg

The biosynthesis of cAMP by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled-receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1 to 7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo . Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5- mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR-Gsα signaling regulating the biosynthesis of cAMP.

### Competing Interest Statement

The authors have declared no competing interest.

A new class of receptors: Lipids regulate mammalian Gsα-stimulated adenylyl cyclase activities via their membrane anchors

lipids-regulate-mammalian-adenylyl-cyclase-activities-via-direct-receptor-interactions


Lipids Regulate Mammalian Adenylyl Cyclase Activities via Direct Receptor Interactions


title_rewrite


젖산탈수소효소 억제는 해당과정, 삼탄소산회로, 산화적 인산화 모두를 억제하는 것으로, 이는 효소 동역학과 열역학적 상호작용을 통해 이루어진다.


Lactate dehydrogenase (LDH) stands at the intersection of pyruvate metabolism. While it is believed that inhibition of LDH redirects pyruvate to mitochondrial metabolism, suppressing glycolysis and boosting oxidative phosphorylation, the mechanism remains largely unexplored. We found that individual LDH A or B knockouts had minimal impact on glycolysis, tricarboxylic acid cycle (TCAC), or oxidative phosphorylation (OXPHOS). However, combining LDH knockout with LDH inhibitor GNE-140 significantly suppressed these processes. Inhibition of LDH led to an increase in free NADH concentration and a decrease in free NAD+ concentration, the reduced free NAD+ concentration inhibited GAPDH, disrupting the balance of glycolytic intermediates, which were linked with thermodynamic shift of the Gibbs free energy of reactions between phosphofructokinase 1 (PFK1) and phosphoglycerate mutase (PGAM) in the glycolytic pathway, favoring their reverse direction. This disrupted glycolysis led to impaired TCAC and mitochondrial respiration due to reduced pyruvate and glutamine carbon influx into TCAC. Under hypoxia, LDH inhibition had a stronger effect, inducing energy crisis, redox imbalance, and cancer cell death. Our study reveals LDH’s intricate control over glycolysis, TCAC, and mitochondrial respiration, highlighting the interplay of enzyme kinetics and thermodynamics in metabolic pathways—a crucial aspect for understanding metabolic regulation.

Impact statement The biochemical mechanism underlying the flux regulation of glycolytic flux by lactate dehydrogenase in cancer cells, which have remained largely unexplored, is deciphered.

Funding information This work has been supported in part by China Natural Science Foundation projects (82073038, 81772947), a key project (2018C03009) funded by Zhejiang Provincial Department of Sciences and Technologies, the Fundamental Research Funds for the Central Universities (226-2024-00062), to X.H.

### Competing Interest Statement

The authors have declared no competing interest.

Elucidating the kinetic and thermodynamic insight into regulation of glycolysis by lactate dehydrogenase and its impact on tricarboxylic acid cycle and oxidative phosphorylation in cancer cells

암세포에서-젖산탈수소효소에-의한-해당과정-조절의-동역학적-및-열역학적-통찰과-삼탄소산회로-및-산화적-인산화에-미치는-영향-규명


암세포에서 젖산탈수소효소에 의한 해당과정 조절의 동역학적 및 열역학적 통찰과 삼탄소산회로 및 산화적 인산화에 미치는 영향 규명



lysostaphin과 LytM은 S. aureus 펩티도글리칸의 D-Ala-Gly 교차결합 부위를 선호적으로 가수분해한다.


Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well-understood, the function, regulation, and mechanism of action of PG hydrolases characterized as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined.

In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

### Competing Interest Statement

The authors have declared no competing interest.

Reassessing the substrate specificities of the major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM

s-aureus-펩티도글리칸-가수분해효소-lysostaphin과-lytm의-기질-특이성-재평가


S. aureus 펩티도글리칸 가수분해효소 lysostaphin과 LytM의 기질 특이성 재평가



The major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM display distinct substrate specificities, with lysostaphin preferring the D-Ala-Gly cross-link in mature peptidoglycan, while LytM can also cleave the D-alanyl-glycine bond in addition to glycyl-glycine bonds.


deciphering-the-substrate-specificities-of-the-major-staphylococcus-aureus-peptidoglycan-hydrolases-lysostaphin-and-lytm


Deciphering the Substrate Specificities of the Major Staphylococcus aureus Peptidoglycan Hydrolases Lysostaphin and LytM



A camelid single-domain antibody (sdAb42) selectively binds and stabilizes the inactive T-state conformation of trypanosomatid pyruvate kinases, thereby inhibiting their enzymatic activity through an allosteric mechanism.


African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth. We propose that these results pinpoint a site of vulnerability on trypanosomatid pyruvate kinases that may be exploited for the design of novel chemotherapeutics.

### Competing Interest Statement

The authors have declared no competing interest.

Allosteric inhibition of trypanosomatid pyruvate kinases by a camelid single-domain antibody

allosteric-inhibition-of-trypanosomatid-pyruvate-kinases-by-a-camelid-single-domain-antibody


Allosteric Inhibition of Trypanosomatid Pyruvate Kinases by a Camelid Single-Domain Antibody



In-gel fluorescence (IGF) detection is a rapid, cost-effective, and quantitative alternative to immunoblotting for visualizing endogenously expressed fluorescent proteins in cell extracts.


The discovery of Green Fluorescent Protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. In this study, we report that various green and red FPs can be maintained in a native, fluorescent form during the entire process of protein sample extraction, incubation with sample buffer, loading and migration on SDS-PAGE with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent to immunoblotting such as transfer onto a membrane and antibody incubations. Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

### Competing Interest Statement

The authors have declared no competing interest.

Direct observation of fluorescent proteins in gels: a rapid cost-efficient, and quantitative alternative to immunoblotting

maintaining-fluorescence-of-endogenously-expressed-proteins-during-sds-page-for-rapid-and-cost-effective-detection


Maintaining Fluorescence of Endogenously Expressed Proteins During SDS-PAGE for Rapid and Cost-Effective Detection



The high-resolution cryo-EM structure of the lysosomal membrane protein HGSNAT in complex with acetyl-CoA provides critical insights into the molecular basis of its N-acetyltransferase activity and the impact of mutations that cause the rare lysosomal storage disorder mucopolysaccharidosis IIIC.


Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N -acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide N -acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in HGSNAT catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.

### Competing Interest Statement

The authors have declared no competing interest.

Structure of the human heparan-α-glucosaminide N-acetyltransferase (HGSNAT)

high-resolution-structure-of-the-lysosomal-heparan-α-glucosaminide-n-acetyltransferase-hgsnat-reveals-insights-into-mucopolysaccharidosis-iiic


High-Resolution Structure of the Lysosomal Heparan-α-Glucosaminide N-Acetyltransferase (HGSNAT) Reveals Insights into Mucopolysaccharidosis IIIC



TTLL10酵素は、既存のモノグリシル化チューブリンに対してのみ高い親和性を示し、ポリグリシル化鎖を伸長する。一方で、ポリグリシル化は逆にTTLL10の微小管への結合を阻害する。また、先行するグルタミル化はTTLL10の微小管結合を促進する。これにより、微小管上の修飾パターンが制御される。


Microtubules in cells have complex and developmentally stereotyped posttranslational modifications that support diverse processes such as cell division, ciliary growth and axonal specification. Glycylation, the addition of glycines, singly (monoglycylation) or in chains (polyglycylation), is primarily found on axonemal microtubules where it functions in cilia maintenance and motility. It is catalyzed by three enzymes in the tubulin tyrosine ligase- like family, TTLL3, 8 and 10. We show that TTLL8 monoglycylates both α- and β-tubulin, unlike TTLL3 which prefers β-tubulin. Microscopy and mass spectrometry show that TTLL10 requires monoglycylation for high affinity microtubule binding and elongates polyglycine chains only from pre-existing glycine branches. Surprisingly, tubulin polyglycylation inhibits TTLL10 recruitment to microtubules proportional with the number of posttranslationally added glycines, suggesting an autonomous mechanism for polyglycine chain length control. In contrast, tubulin glutamylation, which developmentally precedes polyglycylation in cilia, increases TTLL10 recruitment to microtubules, suggesting a mechanism for sequential deposition of tubulin modifications on axonemes. Our work sheds light on how the tubulin code is written by establishing the substrate preference and regulation of TTLL glycylases, and provides a minimal system for generating differentially glycylated microtubules for in vitro analyses of the tubulin code.

### Competing Interest Statement

The authors have declared no competing interest.

The TTLL10 polyglycylase is stimulated by tubulin glutamylation and inhibited by polyglycylation

微小管のポリグリシル化を触媒するttll10酵素は-チューブリンのグルタミル化によって活性化され-ポリグリシル化によって阻害される


微小管のポリグリシル化を触媒するTTLL10酵素は、チューブリンのグルタミル化によって活性化され、ポリグリシル化によって阻害される



DTX3L, a member of the Deltex ubiquitin ligase family, can directly ubiquitinate the 3' hydroxyl group of single-stranded DNA and RNA, expanding the repertoire of non-proteinaceous substrates that undergo ubiquitination.


Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although its N-terminal region contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3’-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3’ overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.

### Competing Interest Statement

DTH is a consultant for Triana Biomedicines. The other authors declare no competing interests.

DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids

dtx3l-ubiquitin-ligase-catalyzes-direct-ubiquitination-of-single-stranded-nucleic-acids


DTX3L Ubiquitin Ligase Catalyzes Direct Ubiquitination of Single-Stranded Nucleic Acids



His-Me 핵산분해효소는 단일 이가 금속 이온과 보존된 히스티딘 잔기를 이용하여 DNA 가수분해를 촉매한다.


Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The superfamily of His-Me nucleases is renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how this single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.

Teaser Soaking crystals of the HNH Cas9 family I-PpoI nuclease reveals how one metal ion and a histidine residue are sufficient for cleaving DNA

### Competing Interest Statement

The authors have declared no competing interest.

Observing one-divalent-metal-ion dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo

단일-이가-금속-이온-의존성-및-히스티딘-촉진-his-me-가족-i-ppoi-핵산분해효소-촉매-과정의-결정-내-관찰


단일 이가 금속 이온 의존성 및 히스티딘 촉진 His-Me 가족 I-PpoI 핵산분해효소 촉매 과정의 결정 내 관찰
