Biology subjects

Peddada, N.

Publications and source records attributed to Peddada, N..

7 recordsLinked to original sources

bioRxiv2026

PPa1 insufficiency drives lysosomal storage disease and inflammatory macrophage expansion in the bone marrow.

Inorganic pyrophosphatase-1 (PPa1) is an essential enzyme proposed to limit accumulation of the ubiquitous metabolic byproduct inorganic pyrophosphate, yet its role beyond a general housekeeping function remains poorly understood. We generated viable hypomorphic alleles of PPa1 through random mutagenesis in mice and unexpectedly found that PPa1 insufficiency causes a lysosomal storage disease of the bone marrow. Mutant mice developed impaired hematopoiesis and defective skeletal mineralization in a hematopoietic-intrinsic manner. The bone marrow was infiltrated by glycolipid-laden macrophages that were marked by high Spp1 and CD14 expression, blunted lysosomal acidification, metabolic stress, and a distinct inflammatory transcriptional program. Consistent with a lysosomal storage defect, PPa1-deficient bone marrow accumulated elevated levels of long-chain glucosyl-sphingolipids and sphingosine. These findings identify PPa1 as a previously unrecognized regulator of lysosomal function in myeloid cells that restrains inflammatory macrophage expansion and prevents bone marrow lysosomal storage pathology.

immunology↗

bioRxiv2025

The Structural Basis of Pacs1-Wdr37 Complex Assembly and Stability

Phosphofurin acidic cluster sorting protein 1 (Pacs1) is a multidomain adaptor proposed to bind transmembrane cargo proteins to facilitate their intracellular trafficking. Pacs1 also forms a complex with WD-repeat protein 37 (Wdr37), which is essential for lymphocyte homeostasis. Despite numerous proposed binding partners, a validated structure for Pacs1-containing protein complexes is lacking. Here, we present the cryo-electron microscopy structure of the Pacs1-Wdr37 complex. Pacs1 binds Wdr37 through a conserved interface within its furin-binding region (FBR), the domain previously linked to cargo recognition. This interaction stabilizes Wdr37 and is critical for the expression of both proteins. A gain-of-function mutation in PACS1 (R203W) causes a highly penetrant neurodevelopmental syndrome. This pathogenic mutation lies on a solvent-exposed surface of the FBR and does not disrupt complex formation. Instead, Pacs1-R203W remains dependent on Wdr37 for stability and its levels can be reduced through targeted Wdr37 proteolytic degradation. Structural homology of the FBR to synaptotagmin C2 domains reveals a previously unrecognized ability of Pacs1 to bind negatively charged phospholipids through a unique positively charged cleft. Together these findings define the structural basis for Pacs1-Wdr37 complex assembly and stability, present potential strategies for Pacs1-mediated neurodevelopmental disease, and suggest novel Pacs1 functions in membrane association.

biochemistry↗

bioRxiv2025

Structural insights into the ubiquitin independent MIDN-proteasome pathway

The protein midnolin (MIDN) augments proteasome activity in lymphocytes, where it is predominantly expressed in adult mice. MIDN binds both to proteasomes and to substrates, but the mechanism by which MIDN facilitates substrate degradation in a ubiquitin-independent manner is unknown. Here, we present cryo-electron microscopy structures of the substrate-engaged, MIDN-bound human proteasome in two conformational states. MIDN induces proteasome conformations similarly to ubiquitinated substrates by using its ubiquitin-like domain to bind to the deubiquitinase RPN11. By simultaneously binding to RPN1 with its C-terminal -helix, MIDN positions its substrate-carrying Catch domain above the proteasome channel through which substrates are translocated before degradation. Our findings suggest that both ubiquitin-like domain and C-terminal -helix must bind to the proteasome for MIDN to stimulate proteasome activity.

biophysics↗

bioRxiv2024

VISUALIZING THE NUCLEATING AND CAPPED STATES OF F-ACTIN BY Ca2+-GELSOLIN: SAXS DATA BASED STRUCTURES OF BINARY AND TERNARY COMPLEXES

Structural insight eludes on how full-length gelsolin depolymerizes and caps F-actin, while the same entity can nucleate polymerization of G-actins. Employing small angle X-ray scattering (SAXS) data analysis, we deciphered these two contrasting assemblies. Mixing Ca2+-gelsolin with F-actin in high salt F-buffer resulted in depolymerization of ordered F-actin rods to smaller sized species which became monodisperse upon dialysis with low salt G-buffer. These entities were the ternary (GA2) and binary (GA) complexes of gelsolin and actin with radius of gyration and maximum linear dimension of 4.55 and 4.68 nm, and 15 and 16 nm, respectively. In contrast, upon mixing G-actin with Ca2+-gelsolin in G-buffer, rapid association of higher order species started. Using size exclusion chromatography in-line with SAXS, we confirmed that initially GA and GA2 species are formed as seen upon depolymerization of F-actin, followed by dialysis. Interestingly, while GA2 could seed formation of native-like F-actin in both G- and F-buffer, GA failed in G-buffer. Thus, GA2 and GA are the central species formed via depolymerization or towards nucleation. SAXS profile referenced modeling revealed that: 1) in GA, actin is bound to the C-terminal half of gelsolin, and 2) in GA2, second actin binds to the open N-terminal half accompanied by dramatic rearrangements across gelsolins g1-g2 and g3-g4 linkers. Importantly, first structural insight is provided into the two probable models for GA2 with two actins in parallel, but differentially stacked: one in polymerization competent, and other in incompetent manner, suggesting latter to represent capped state along with the inert GA. TOC Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/584204v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@9b7ab4org.highwire.dtl.DTLVardef@194d85aorg.highwire.dtl.DTLVardef@108151dorg.highwire.dtl.DTLVardef@1a24e88_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIOrderly decrement in the length of F-actin by Ca2+-gelsolin was tracked by SAXS. C_LIO_LIResidual re-association in 1:2 ratio in F-buffer was quenched by dialysis in G-buffer. C_LIO_LIIdentical GA and GA2 entities formed upon mixing F- or G-actin with Ca2+-gelsolin. C_LIO_LIModels of nucleation competent, GA2 showed differential stacking of two G-actins. C_LIO_LIN-terminal half of gelsolin reposition as GA2 changes to or from GA, the capped state. C_LI

biophysics↗

bioRxiv2024

SAXS DATA BASED GLYCOSYLATED MODELS OF HUMAN ALPHA-1-ACID GLYCORPROTEIN, A KEY PLAYER IN HEALTH, DISEASE AND DRUG CIRCULATION

Plasma Alpha-1-glycoprotein (AGP) binds diverse drugs, its isoforms and their levels vary significantly in acute phases of health. Relative binding of drugs to AGP and albumin has been used to model their release profiles, structural insights on glycosylated form of AGP will certainly improve estimations. Main challenges are: 1)heavy glycosylation of AGP (~50% of its mass), 2) isoforms of primary structure co-exist, and 3) extreme heterogeneity in the glycan motifs have been reported. Our small angle X-ray scattering (SAXS) data on plasma extracted AGP showed interparticulate effect from 283-313 K which disappeared irreversibly upon further heating to 343K. Using ALPHAFOLD2 server, the protein only portion could be modelled but its theoretical SAXS profile did not match acquired experimental data. Using mass spectra-based information, we attached glycan motifs at different known sites to compute four models of fully glycosylated AGP. Importantly, calculated SAXS profiles of our glycosylated models agreed well with the experimental data. Docking runs revealed that in silico interaction of different drugs when varied using unglycosylated vs. glycosylated model as receptor. Finally, we propose that our SAXS based models of glycoprotein are better representation of the molecule and should be considered for structure-based analysis and/or estimations.

biophysics↗

bioRxiv2023

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

biophysics↗

bioRxiv2022

Sequence-directed RNA remodeling within a topologically complex RNP substrate

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes is at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-directed remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize a post-catalytic, high-energy intermediate that drives the organization of the root helix structure within rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

biophysics↗