We uncovered a novel pathway of N-acetylglucosamine (GlcNAc) metabolism for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis via Ngk1 kinase in Saccharomyces cerevisiae. GlcNAc phosphorylation by Ngk1 promotes UDP-GlcNAc synthesis and compensates for the hexosamine pathway, a known pathway for UDP-GlcNAc synthesis. The increased synthesis of UDP-GlcNAc by Ngk1 enhances chitin production.

N-acetylglucosamine (GlcNAc) is an important structural component of the cell wall chitin, N-glycans, glycolipids, and GPI-anchors in eukaryotes. GlcNAc kinase phosphorylates GlcNAc into GlcNAc-6-phosphate, a precursor of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that serves as a substrate for glycan synthesis. Although GlcNAc kinase is found widely in organisms ranging from microorganisms to mammals, it has never been found in the model yeast Saccharomyces cerevisiae. Here, we demonstrate the presence of GlcNAc metabolism for UDP-GlcNAc biosynthesis in S. cerevisiae through Ngk1, a GlcNAc kinase we discovered previously. The overexpression or deletion of Ngk1 in the presence of GlcNAc affected the amount of both UDP-GlcNAc and chitin, suggesting that GlcNAc metabolism via Ngk1 promotes UDP-GlcNAc synthesis. Our data suggest that the Ngk1-mediated GlcNAc metabolism compensates for the hexosamine pathway, a known pathway for UDP-GlcNAc synthesis.

We investigated the role of the mRNA decay factor Protein Associated with Topoisomerase II (PAT1) by generating multiple mutants of pat1 and its homologs, path1 and path2. Through detailed analysis of their developmental phenotypes and RNA sequencing, we concluded that PAT1 plays a crucial role in leaf patterning. Moreover, it operates in conjunction with the other two PATs redundantly throughout plant development.

Evolutionarily conserved protein associated with topoisomerase II (PAT1) proteins activate mRNA decay through binding mRNA and recruiting decapping factors to optimize posttranscriptional reprogramming. Here, we generated multiple mutants of pat1, pat1 homolog 1 (path1), and pat1 homolog 2 (path2) and discovered that pat triple mutants exhibit extremely stunted growth and all mutants with pat1 exhibit leaf serration while mutants with pat1 and path1 display short petioles. All three PATs can be found localized to processing bodies and all PATs can target ASYMMETRIC LEAVES 2-LIKE 9 transcripts for decay to finely regulate apical hook and lateral root development. In conclusion, PATs exhibit both specific and redundant functions during different plant growth stages and our observations underpin the selective regulation of the mRNA decay machinery for proper development.

When bound to toxic or non-toxic ligands, aryl hydrocarbon receptors (AhRs) are activated and nuclear translocation occurs; AhRs are bound to molecular chaperone complexes, AhR-HSP90-XAP2-p23 for toxic ligands, whereas for non-toxic ligands AhR-HSP90- XAP2 for non-toxic ligands. Toxic and non-toxic ligand selectivity of AhR depends on the components of the molecular chaperone complex.

The aryl hydrocarbon receptor (AhR) forms a complex with the HSP90-XAP2-p23 molecular chaperone when the cells are exposed to toxic compounds. Recently, 1,4-dihydroxy-2-naphthoic acid (DHNA) was reported to be an AhR ligand. Here, we investigated the components of the molecular chaperone complex when DHNA binds to AhR. Proteins eluted from the 3-Methylcolanthrene-affinity column were AhR-HSP90-XAP2-p23 complex. The AhR-molecular chaperone complex did not contain p23 in the eluents from the DHNA-affinity column. In 3-MC-treated cells, AhR formed a complex with HSP90-XAP2-p23 and nuclear translocation occurred within 30 min, while in DHNA-treated cells, AhR formed a complex with AhR-HSP90-XAP2, and translocation was slow from 60 min. Thus, the AhR activation mechanism may differ when DHNA is the ligand compared to toxic ligands.

Contrary to previous predictions, we provide evidence that the small GTPase κB-Ras possesses intrinsic hydrolytic activity. However, low nucleotide affinity leads to fast nucleotide exchange and renders κB-Ras constitutively GTP-bound in cells. We characterize κB-Ras mutations occurring in tumors and define that nucleotide binding supports protein stability but is not required for a constitutive noneffector interaction with RalGAP complexes.

κB-Ras (NF-κB inhibitor-interacting Ras-like protein) GTPases are small Ras-like GTPases but harbor interesting differences in important sequence motifs. They act in a tumor-suppressive manner as negative regulators of Ral (Ras-like) GTPase and NF-κB signaling, but little is known about their mode of function. Here, we demonstrate that, in contrast to predictions based on primary structure, κB-Ras GTPases possess hydrolytic activity. Combined with low nucleotide affinity, this renders them fast-cycling GTPases that are predominantly GTP-bound in cells. We characterize the impact of κB-Ras mutations occurring in tumors and demonstrate that nucleotide binding affects κB-Ras stability but is not strictly required for RalGAP (Ral GTPase-activating protein) binding. This demonstrates that κB-Ras control of RalGAP/Ral signaling occurs in a nucleotide-binding- and switch-independent fashion.

The authors find that, in normal hematopoiesis, the DMT1/SLC11A2 nonIRE isoform controls the hematopoietic stem cell pool by dictating differentiation of the myeloid and B cell lymphoid lineages while suppressing the production of platelets via Notch/Myc pathway regulation. In TLX1-defective leukemia, DMT1 nonIRE boosts NOTCH pathway activity, known to be responsible for unlimited proliferation of leukemic cells.

Natural resistance-associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron-response element (nonIRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non-canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non-canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T-cell leukemia homeobox protein 1 (TLX1)-defective leukemia. This work sets the stage for future investigation using a multiple-hit T-cell acute lymphoblastic leukemia (T-ALL) model to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression.

Here, we detected stem cell memory T cells (TSCMs) in chronic hepatitis B virus (HBV)-infected patients, using HBV core and polymerase peptide HLA tetramers. When these TSCMs were transferred into the human hepatocyte-transplanted, HBV-infected TK-NOG mouse, they differentiated into cytotoxic T lymphocytes, produced interferon gamma and interleukin-2, and developed histologically proven hepatitis. Furthermore, HBV DNA and human albumin in mouse serum declined and human hepatocytes were eliminated in the mouse model.

Chronic infection with the hepatitis B virus (HBV) induces progressive hepatic impairment. Achieving complete eradication of the virus remains a formidable challenge. Cytotoxic T lymphocytes, specific to viral antigens, either exhibit a numerical deficiency or succumb to an exhausted state in individuals chronically afflicted with HBV. The comprehension of the genesis and dissemination of stem cell memory T cells (TSCMs) targeting HBV remains inadequately elucidated. We identified TSCMs in subjects with chronic HBV infection and scrutinized their efficacy in a murine model with human hepatocyte transplants, specifically the TK-NOG mice. TSCMs were discerned in all subjects under examination. Introduction of TSCMs into the HBV mouse model precipitated a severe necro-inflammatory response, resulting in the elimination of human hepatocytes. TSCMs may constitute a valuable tool in the pursuit of a remedial therapy for HBV infection.

The region containing residues I649 − L659 of the epidermal growth factor receptor (EGFR) juxtamembrane domain (JM) specifically interacts with PI(4,5)P₂-or phosphatidylserine-containing membranes, suggesting that membrane binding may affect JM dimerization and, therefore, regulate EGFR kinase activation.

Overactivation of the epidermal growth factor receptor (EGFR) is critical for the development of multiple cancers. Previous studies have shown that the cell membrane is a key regulator of EGFR kinase activity through its interaction with the EGFR juxtamembrane domain (JM). However, the lipid recognition specificity of EGFR-JM and its interaction details remain unclear. Using lipid strip and liposome pulldown assays, we showed that EGFR-JM could specifically interact with PI(4,5)P₂-or phosphatidylserine-containing membranes. We further characterized the JM–membrane interaction using NMR-titration-based chemical shift perturbation and paramagnetic relaxation enhancement analyses, and found that residues I649 − L659 comprised the membrane-binding site. Furthermore, the membrane-binding region contains the predicted dimerization motif of JM, ⁶⁵⁵LRRLL⁶⁵⁹, suggesting that membrane binding may affect JM dimerization and, therefore, regulate kinase activation.

X-ray crystallography was used to determine an active-like dimer of the ligand-binding domain of GluK2, locating the positive allosteric modulator BPAM344 within the dimeric interface. Selected residues T535 and Q786 differing between GluK2 and GluA2 were investigated with calcium-sensitive fluorescence-based assays on transiently transfected cells. A three-fold increase in BPAM344 potency occurred with the double mutant T535S-Q786S in GluK2(Q).

Kainate receptors belong to the family of ionotropic glutamate receptors and contribute to the majority of fast excitatory neurotransmission. Consequently, they also play a role in brain diseases. Therefore, understanding how these receptors can be modulated is of importance. Our study provides a crystal structure of the dimeric ligand-binding domain of the kainate receptor GluK2 in complex with L-glutamate and the small-molecule positive allosteric modulator, BPAM344, in an active-like conformation. The role of Thr535 and Gln786 in modulating GluK2 by BPAM344 was investigated using a calcium-sensitive fluorescence-based assay on transiently transfected cells expressing GluK2 and mutants hereof. This study may aid in the design of compounds targeting kainate receptors, expanding their potential as targets for the treatment of brain diseases.

Protein components of the germ granules, Aubergine (Aub) and Pyruvate Kinase (PyK), use different modes to associate with Tudor (Tud) protein, which contains 11 Tud domains. Aub binds to a single Tud domain and PyK requires two Tud domains. Aub and PyK compete for binding to Tud in vitro and form separate clusters within a granule in vivo, thereby minimizing the competition.

Membraneless organelles are RNA–protein assemblies which have been implicated in post-transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain-containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi-domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule.

The circumsporozoite protein of Plasmodium falciparum (Pf-CSP) contains two nuclear localization signals (NLSs). The lysine and arginine residues in both NLSs are essential for Pf-CSP interaction with importin-α, aiding Pf-CSP's nuclear translocation. Both NLSs are vital for nuclear localization, with individual NLSs resulting in weaker localization. Pf-CSP also contains a distinct nuclear export signal motif that coordinates the protein nucleocytoplasmic shuttling mechanisms.

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.