Danish Khan

All Publications

• ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors. eLife Alford, B. D., Tassoni-Tsuchida, E., Khan, D., Work, J. J., Valiant, G., Brandman, O. 2021; 10

  Abstract

  Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.

  View details for DOI 10.7554/eLife.57376

  View details for PubMedID 34223816

• A Sec14-like phosphatidylinositol transfer protein paralog defines a novel class of heme-binding proteins ELIFE Khan, D., Lee, D., Gulten, G., Aggarwal, A., Wofford, J., Krieger, I., Tripathi, A., Patrick, J. W., Eckert, D. M., Laganowsky, A., Sacchettini, J., Lindahl, P., Bankaitis, V. A. 2020; 9

  Abstract

  Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIII hemoprotein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.

  View details for DOI 10.7554/eLife.57081

  View details for Web of Science ID 000567780500001

  View details for PubMedID 32780017

  View details for PubMedCentralID PMC7462610

• Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle BIOPHYSICAL JOURNAL Sugiura, T., Takahashi, C., Chuma, Y., Fukuda, M., Yamada, M., Yoshida, U., Nakao, H., Ikeda, K., Khan, D., Nile, A. H., Bankaitis, V. A., Nakano, M. 2019; 116 (1): 92–103

  Abstract

  Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.

  View details for DOI 10.1016/j.bpj.2018.11.3131

  View details for Web of Science ID 000455089100012

  View details for PubMedID 30580923

  View details for PubMedCentralID PMC6342728

• Target Identification and Mechanism of Action of Picolinamide and Benzamide Chemotypes with Antifungal Properties CELL CHEMICAL BIOLOGY Pries, V., Noecker, C., Khan, D., Johnen, P., Hong, Z., Tripathi, A., Keller, A., Fitz, M., Perruccio, F., Filipuzzi, I., Thavam, S., Aust, T., Riedl, R., Ziegler, S., Bono, F., Schaaf, G., Bankaitis, V. A., Waldmann, H., Hoepfner, D. 2018; 25 (3): 279-+

  Abstract

  Invasive fungal infections are accompanied by high mortality rates that range up to 90%. At present, only three different compound classes are available for use in the clinic, and these often suffer from low bioavailability, toxicity, and drug resistance. These issues emphasize an urgent need for novel antifungal agents. Herein, we report the identification of chemically versatile benzamide and picolinamide scaffolds with antifungal properties. Chemogenomic profiling and biochemical assays with purified protein identified Sec14p, the major phosphatidylinositol/phosphatidylcholine transfer protein in Saccharomyces cerevisiae, as the sole essential target for these compounds. A functional variomics screen identified resistance-conferring residues that localized to the lipid-binding pocket of Sec14p. Determination of the X-ray co-crystal structure of a Sec14p-compound complex confirmed binding in this cavity and rationalized both the resistance-conferring residues and the observed structure-activity relationships. Taken together, these findings open new avenues for rational compound optimization and development of novel antifungal agents.

  View details for DOI 10.1016/j.chembiol.2017.12.007

  View details for Web of Science ID 000427600400008

  View details for PubMedID 29307839

  View details for PubMedCentralID PMC5856591

• Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors JOURNAL OF LIPID RESEARCH Khan, D., McGrath, K. R., Dorosheva, O., Bankaitis, V. A., Tripathi, A. 2016; 57 (4): 650–62

  Abstract

  Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.

  View details for DOI 10.1194/jlr.M066381

  View details for Web of Science ID 000373924600014

  View details for PubMedID 26921357

  View details for PubMedCentralID PMC4808773

• Phosphatidylinositol transfer proteins and instructive regulation of lipid kinase biology BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS Grabon, A., Khan, D., Bankaitis, V. A. 2015; 1851 (6): 724–35

  Abstract

  Phosphatidylinositol is a metabolic precursor of phosphoinositides and soluble inositol phosphates. Both sets of molecules represent versatile intracellular chemical signals in eukaryotes. While much effort has been invested in understanding the enzymes that produce and consume these molecules, central aspects for how phosphoinositide production is controlled and functionally partitioned remain unresolved and largely unappreciated. It is in this regard that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are emerging as central regulators of the functional channeling of phosphoinositide pools produced on demand for specific signaling purposes. The physiological significance of these proteins is amply demonstrated by the consequences that accompany deficits in individual PITPs. Although the biological problem is fascinating, and of direct relevance to disease, PITPs remain largely uncharacterized. Herein, we discuss our perspectives regarding what is known about how PITPs work as molecules, and highlight progress in our understanding of how PITPs are integrated into cellular physiology. This article is part of a Special Issue entitled Phosphoinositides.

  View details for DOI 10.1016/j.bbalip.2014.12.011

  View details for Web of Science ID 000353095800004

  View details for PubMedID 25592381

  View details for PubMedCentralID PMC5221696

• Stalled translation by mitochondrial stress upregulates a CNOT4-ZNF598 ribosomal quality control pathway important for tissue homeostasis. Nature communications Geng, J., Li, S., Li, Y., Wu, Z., Bhurtel, S., Rimal, S., Khan, D., Ohja, R., Brandman, O., Lu, B. 2024; 15 (1): 1637

  Abstract

  Translational control exerts immediate effect on the composition, abundance, and integrity of the proteome. Ribosome-associated quality control (RQC) handles ribosomes stalled at the elongation and termination steps of translation, with ZNF598 in mammals and Hel2 in yeast serving as key sensors of translation stalling and coordinators of downstream resolution of collided ribosomes, termination of stalled translation, and removal of faulty translation products. The physiological regulation of RQC in general and ZNF598 in particular in multicellular settings is underexplored. Here we show that ZNF598 undergoes regulatory K63-linked ubiquitination in a CNOT4-dependent manner and is upregulated upon mitochondrial stresses in mammalian cells and Drosophila. ZNF598 promotes resolution of stalled ribosomes and protects against mitochondrial stress in a ubiquitination-dependent fashion. In Drosophila models of neurodegenerative diseases and patient cells, ZNF598 overexpression aborts stalled translation of mitochondrial outer membrane-associated mRNAs, removes faulty translation products causal of disease, and improves mitochondrial and tissue health. These results shed lights on the regulation of ZNF598 and its functional role in mitochondrial and tissue homeostasis.

  View details for DOI 10.1038/s41467-024-45525-3

  View details for PubMedID 38388640

• Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity. The Journal of biological chemistry Chen, X., Poudel, L., Hong, Z., Johnen, P., Katti, S., Tripathi, A., Nile, A. H., Green, S. M., Khan, D., Schaaf, G., Bono, F., Bankaitis, V. A., Igumenova, T. I. 2023; 299 (2): 102861

  Abstract

  Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

  View details for DOI 10.1016/j.jbc.2022.102861

  View details for PubMedID 36603766

• Sis1 delivers the State of the Union. The Journal of cell biology Khan, D. n., Brandman, O. n. 2021; 220 (1)

  Abstract

  The heat shock response (HSR) is a gene expression program that protects cells from heat and proteotoxic stressors. In this issue, Feder et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202005165) show that subcellular relocalization of the cochaperone Sis1 drives the HSR by de-suppressing the transcription factor Hsf1.

  View details for DOI 10.1083/jcb.202011093

  View details for PubMedID 33332552