Biology subjects

Dalby, K. N.

Publications and source records attributed to Dalby, K. N..

6 recordsLinked to original sources

bioRxiv2025

The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the -kinase family, modulates translational rates by phosphorylating eEF-2, a GTPase that facilitates the translocation of the nascent chain on the ribosome during the elongation phase of protein synthesis. eEF-2K is regulated by diverse cellular cues, many of which sensitize it to the Ca2+-effector protein calmodulin (CaM). CaM, which binds and allosterically activates eEF-2K in the presence of Ca2+, contains two structural "lobes," each with a pair of Ca2+-binding EF-hands. Using kinetic analysis, we demonstrate that the isolated C-terminal lobe of CaM (CaMC) is sufficient to engage and fully activate eEF-2K in a Ca2+-dependent fashion. Genetically fusing CaMC to the N-terminus of eEF-2K, upstream of its critical CaM-targeting motif (CTM) via a flexible 2-glycine linker, results in a chimeric species (C-LiNK) that is constitutively active independent of external CaM and Ca2+. A structure of the C-LiNK functional core reveals no significant deviation in the overall conformations of the interacting modules and orientations of key catalytic-site residues relative to the heterodimeric complex between full-length CaM and eEF-2K. These observations demonstrate that, in contrast to other CaM-regulated kinases, CaMC alone is sufficient to activate eEF-2K fully. The proximity effect of CaMC in the context of C-LiNK removes the requirement for external Ca2+, whose apparent role is to enhance the CaM-affinity of eEF-2K and drive kinase activation. The responsiveness of eEF-2K to regulatory stimuli in cells appears to be lost in C-LiNK, presumably due to its permanently "on" state.

biochemistry↗

bioRxiv2023

Transcription Factor EB (TFEB) activity increases resistance of TNBC stem cells to metabolic stress

Breast Cancer Stem Cells (CSCs) are difficult to therapeutically target, but continued efforts are critical given their contribution to tumor heterogeneity and treatment resistance in Triple-Negative Breast Cancer (TNBC). CSC properties are influenced by metabolic stress, but specific mechanisms are lacking for effective drug intervention. Our previous work on TFEB suggested a key function in CSC metabolism. Indeed, TFEB knockdown (KD) inhibited mammosphere formation in vitro and tumor initiation/growth in vivo. These phenotypic effects were accompanied by a decline in CD44high/CD24low cells. Glycolysis inhibitor 2-deoxy-D-glucose (2-DG) induced TFEB nuclear translocation, indicative of TFEB transcriptional activity. TFEB KD blunted, whereas TFEB (S142A) augmented 2-DG-driven UPR mediators, notably BiP/HSPA5 and CHOP. Like TFEB KD, silencing BiP/HSPA5 inhibited CSC self-renewal, suggesting that TFEB augments UPR-related survival. Further studies showed that TFEB KD attenuated 2-DG-directed autophagy, suggesting a mechanism whereby TFEB protects CSCs against 2-DG-induced stress. Our data indicate that TFEB modulates CSC metabolic stress response via autophagy and UPR. These findings reveal the novel role of TFEB in regulating CSCs during metabolic stress in TNBC. Financial SupportThis work was supported by CPRIT Grant RR160093 (to S.G. Eckhardt), CPRIT Grant RP210088 (to K.N. Dalby), UT College of Pharmacy Non-discretionary Funds (to C. Van Den Berg), and UT Graduate Continuing Fellowship (to M. Soleimani).

cancer biology↗

bioRxiv2023

Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control

p38 mitogen-activated protein kinases (MAPKs) regulate early endocytic trafficking, but their effects on late endocytic trafficking remain unclear. Herein, we report that the pyridinyl imidazole p38 MAPK inhibitors, SB203580 and SB202190, induce a rapid but reversible Rab7-dependent accumulation of large cytoplasmic vacuoles. While SB203580 did not induce canonical autophagy, phosphatidylinositol 3-phosphate [PI(3)P] accumulated on vacuole membranes, and inhibition of the class III PI3-kinase (PIK3C3/VPS34) suppressed vacuolation. Ultimately, vacuolation resulted from the fusion of ER/Golgi-derived membrane vesicles with late endosomes and lysosomes (LELs), combined with an osmotic imbalance in LELs that led to severe swelling and a decrease in LEL fission. Since PIKfyve inhibitors induce a similar phenotype by preventing the conversion of PI(3)P to PI(3,5)P2, we performed in vitro kinase assays and found that PIKfyve activity was unexpectedly inhibited by SB203580 and SB202190, corresponding to losses in endogenous PI(3,5)P2 levels in treated cells. However, vacuolation was not entirely due to off-target inhibition of PIKfyve by SB203580, as a drug-resistant p38 mutant suppressed vacuolation. Moreover, genetic deletion of both p38 and p38{beta} rendered cells dramatically more sensitive to PIKfyve inhibitors, including YM201636 and apilimod. In subsequent washout experiments, the rate of vacuole dissolution upon the removal of apilimod was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, p38 MAPKs act epistatically to PIKfyve to promote LEL fission; and pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs.

cell biology↗

bioRxiv2022

Biomechanical dependence of SARS-CoV-2 infections

Older people have been disproportionately vulnerable to the current SARS-CoV-2 pandemic, with an increased risk of severe complications and death compared to other age groups. A mix of underlying factors has been speculated to give rise to this differential infection outcome, including changes in lung physiology, weakened immunity, and severe immune response. Our study focuses on the impact of biomechanical changes in lungs that occur as individuals age, i.e., the stiffening of the lung parenchyma and increased matrix fiber density. We used hydrogels with an elastic modulus of 0.2 and 50 kPa and conventional tissue culture surfaces to investigate how infection rate changes with parenchymal tissue stiffness in lung epithelial cells challenged with SARS-CoV-2 Spike (S) protein pseudotyped lentiviruses. Further, we employed electrospun fiber matrices to isolate the effect of matrix density. Given the recent data highlighting the importance of alternative virulent strains, we included both the native strain identified in early 2020 and an early S protein variant (D614G) that was shown to increase the viral infectivity markedly. Our results show that cells on softer and sparser scaffolds, closer resembling younger lungs, exhibit higher infection rates by the WT and D614G variant. This suggests that natural changes in lung biomechanics do not increase the propensity for SARS-CoV-2 infection and that other factors, such as a weaker immune system, may contribute to increased disease burden in the elderly.

bioengineering↗

bioRxiv2022

Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

Translation is a highly energy consumptive process1 tightly regulated for optimal protein quality2 and adaptation to energy and nutrient availability. A key facilitator of this process is the -kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis3,4. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process5 mechanistically distinct from other calmodulin-dependent kinases6. Here we resolve the atomic details of this mechanism through a 2.3 [A] crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a "spine" that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

biochemistry↗

bioRxiv2021

Rapid characterization of spike variants via mammalian cell surface display

The SARS-CoV-2 spike (S) protein is a critical component of subunit vaccines and a target for neutralizing antibodies. Spike is also undergoing immunogenic selection with clinical variants that increase infectivity and partially escape convalescent plasma. Here, we describe spike display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed [~]200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by thirteen neutralizing antibodies (nAbs). An alanine scan of all five N-terminal domain (NTD) loops highlights a public class of epitopes in the N1, N3, and N5 loops that are recognized by most of the NTD-binding nAbs. Some clinical NTD substitutions abrogate binding to these epitopes but are circulating at low frequencies around the globe. NTD mutations in variants of concern B.1.1.7 (United Kingdom), B.1.351 (South Africa), B.1.1.248 (Brazil), and B.1.427/B.1.429 (California) impact spike expression and escape most NTD-targeting nAbs. However, two classes of NTD nAbs still bind B.1.1.7 spikes and neutralize in pseudoviral assays. B.1.351 and B.1.1.248 include compensatory mutations that either increase spike expression or increase ACE2 binding affinity. Finally, B.1.351 and B.1.1.248 completely escape a potent ACE2 peptide mimic. We anticipate that spike display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.

biochemistry↗