Abstract WNK (With No lysine K) protein kinases gained early attention because gain-of-function mutations in genes encoding two of them cause hypertension. They differ from other kinases due to the unusual location of the catalytic lysine, hence the name WNK. These kinases control many aspects of ion transport in the kidney and are essential for development and maintenance of the cardiovascular system. In the present review, we describe WNK proteins and selected substrates, in particular, the kinases oxidative stress-responsive kinase 1 and SPS1-related proline/alanine-rich kinase, and their regulation. We spotlight findings that emphasize the broad relevance of WNKs in normal physiology and disease. Our overview focuses on basic aspects of ion transport that occur in many cell types and tissues, functions in the endothelial system, interactions with insulin-dependent and metabolic pathways, the importance in the brain in part through controlling chloride ion balance, and the whirlwind of findings in cancer that reveal how WNK signaling can be repurposed. Other actions that may be of equal importance, e.g., influences on the immune system, are touched on only briefly. We also note a few genome-wide association studies that have uncovered multiple SNPs in genes encoding WNKs and downstream effectors in an array of diseases, in anticipation of more to come in the future.
Abstract Biomolecular condensates, formed through phase separation of multivalent biomolecules, play essential roles in cellular processes such as stress response, RNA processing, transcriptional control, and genome organization. Nucleic acids are increasingly recognized as important drivers and regulators of condensate formation and dynamics. This review examines recent advances in coarse-grained molecular simulations of dynamic nucleic acids and their phase transitions. We survey established computational frameworks validated against experimental data and discuss emerging applications that address increasingly complex systems. Existing studies reveal that base-mediated interactions emerge as dominant drivers in homotypic nucleic acid condensation and that simulation resolution fundamentally determines which physical phenomena are accessible and observable. We underscore that different modeling approaches at various resolutions provide complementary insights into nucleic acid condensation mechanisms and the driving forces. We emphasize the opportunities and challenges in extending these frameworks to capture the complexity of nucleic acid-mediated phase separation in biological contexts, while identifying important gaps in current modeling capabilities. Overall, molecular simulations at multiple scales have proven to be powerful tools for elucidating the mechanistic principles underlying nucleic acid condensates and their cellular functions.
Abstract The classical paradigm of the sphingolipid rheostat—balancing pro-apoptotic ceramide against pro-survival sphingosine-1-phosphate—has evolved into a complex signaling landscape where N-acyl chain length serves as the fundamental determinant of biological outcomes. This perspective explores the pivotal role of ceramide synthase 2 (CerS2) in synthesizing very-long-chain (VLC) ceramides (C22–C26) and its function as a central regulator of cellular stress responses. While CerS2 typically acts as a tumor suppressor, its activity is frequently attenuated in aggressive malignancies through alternative splicing or protein inhibition. The emergence of DH20931, a first-in-class small-molecule CerS2 agonist, is discussed as a paradigm-shifting tool for re-engaging suppressed apoptotic pathways. The present paper outlines a sequential-mechanism model for pharmacological CerS2 activation, which drives lipotoxic endoplasmic reticulum (ER) stress via the PERK-ATF4-CHOP axis and tightens the ER-mitochondria synapse through direct IP3R1 interaction. The latter mechanism facilitates lethal calcium influx, triggering the mitochondrial caspase-dependent apoptotic pathway. Furthermore, the preclinical performance of DH20931 is evaluated in models of triple-negative breast cancer (TNBC) relative to standard-of-care therapeutics. The conclusion examines the future of CerS2 as a major druggable target, emphasizing the need for companion diagnostics to identify patients most likely to benefit from agonist therapy based on specific splice variants and metabolic profiles. Combining CerS2 activation with existing chemo- or immunotherapies represents a promising synergistic strategy for treating resistant solid tumors.
Deltex E3 ligases such as DTX1 and DTX2 are known to bind to ADP-ribosylated substrates and consequently ubiquitinate them. However, little is known about the role of the deltex E3 ligase DTX4 in substrate binding and ubiquitination. Here, we present the crystal structure of the DTX4 RING-DTC domains in complex with ADP-ribose to a final resolution of 2.8 Å and uncover a unique domain swap within the RING domain. Structural comparison with the closely related RING-DTC domains of DTX1 and DTX2 reveals similarities but also differences in the mode of ligand binding and the number and type of conserved residues. Biophysical assays to assess the strength of binding to nucleotide units of poly-ADP-ribose (PAR) demonstrate a preference for binding to ADP-ribose, strengthening existing evidence for PAR binding. Collectively, the structural and binding data offer insights into the active structure of DTX4 and its interaction with nucleotide units of PAR.
Methyl-CpG-binding domain protein 4 (MBD4) is a base excision repair glycosylase that removes the thymine of a T:G mispair. If left unrepaired, these mismatches can cause C>T transitions. Human MBD4 uses an arginine finger, R468, to flip the mispaired thymine into its active site and to stabilize the orphaned guanine. MBD4 also possesses a second highly conserved arginine, R512, in close proximity to R468, whose role remains unknown despite an R512W mutation catalogued in colorectal cancers and an R512Q mutation reported in Myelodysplastic Syndromes. Here, we investigate the structural and functional significance of R512 using MBD4 variants R512A, R512K, R512Q, and R512W. We find that the native hydrogen-bonding network mediated by R512 is critical for active-site stability, and that R512K is the only variant to retain catalytic activity. This mutant preserves the wild-type rate of base excision despite a reduced affinity for the DNA substrate. Structural characterization of R512K MBD4 bound to T:G-mismatched DNA reveals an ordered water molecule in this pocket. This water molecule, along with the lysine amino group, retain 6 of the 7 native R512 hydrogen bonds, thereby preserving the local architecture of the enzyme. Comparative structural modeling of the catalytically inactive R512A, R512Q, and R512W variants suggests that loss of localized positive charge combined with disruption of the hydrogen-bonding network accounts for their inactivity. Together, these findings establish an essential role for R512 in catalytic activity and suggest how mutation of this residue can disrupt the BER pathway and contribute to tumorigenesis.
