Biology subjects

Diedrich, J. K.

Publications and source records attributed to Diedrich, J. K..

At least 19 recordsLinked to original sources

bioRxiv2026

dnoise: Fast Native Data Reduction for Bruker timsTOF

Bruker timsTOF acquisitions produce dense native .d files whose storage, transfer, and archival become substantial at high throughput. We present dnoise, an open-source Rust tool that removes points directly from timsTOF frames and writes a native-compatible .d directory. dnoise retains ions that form coherent streaks across the ion-mobility dimension and applies acquisition-aware gates to signal that cannot be selected for fragmentation. On a three-species benchmark spanning ddaPASEF and diaPASEF at 5- and 15-minute gradients, default MS1-only denoising reduced the frame binary by 35 to 53%. Label-free quantification accuracy was preserved in both modes. ddaPASEF peptide-spectrum-match, peptide, and protein-group counts were unchanged, as expected with the searched MS/MS spectra untouched, and diaPASEF precursor and protein-group counts changed only slightly. Every tested processing run completed in 69 seconds or less on the benchmark workstation. Optional MS/MS denoising produced greater reduction but sacrificed several percent of identifications. Thus, a substantial fraction of native timsTOF frame data can be removed with little analytical change.

bioinformatics↗

bioRxiv2026

Rapid Histone Post-Translational Modification Analysis Using Alternative Proteases and Tandem Mass Tags

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease, yet mass spectrometry-based histone PTM analysis remains constrained by inefficient sample preparation workflows. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation to hours while improving PTM coverage and quantitative accuracy. Systematic evaluation of Arg-C Ultra and a recombinant (r)-Chymotrypsin protease under varied conditions, including standard derivatization with propionic anhydride and tandem mass tag (TMT) labeling, demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM that exceeds Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMTs tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 58 succinylation and 31 glutarylation sites - a dark epigenome largely undetected by propionylation-based methods. Complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. In HEK293T cells treated with the pan-sirtuin inhibitor nicotinamide, RIPUP quantified 112 statistically significant peptidoforms (adj p < 0.05), predominantly increasing with NAM dose (88 up, 24 down). Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identified >200 PTMs including H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A K118/K119 ubiquitination. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.

biochemistry↗

bioRxiv2025

Redox regulation of neuroinflammatory pathways contributes to damage in Alzheimers disease brain

The mechanism(s) whereby redox stress mediates aberrant immune signaling in age-related neurological disorders remains largely unknown. Normally, the innate immune system mounts a robust response to infectious stimuli. However, unintentional activation by host-derived factors, such as aggregated proteins associated with neurodegenerative disorders or by cytoplasmic genomic or mitochondrial DNA, can elicit aberrant immune responses. One such immune response is represented by the cytosolic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. Using redox chemical biology and mass spectrometry approaches, we identified S-nitrosylation of STING cysteine 148 as a novel posttranslational redox modification underlying aberrant type 1 interferon signaling in Alzheimers disease (AD). Critically, we observed S-nitrosylated STING (SNO-STING) in postmortem human AD brains, in hiPSC-derived microglia (hiMG) exposed to amyloid-{beta} (A{beta})/-synuclein (Syn) aggregates, and in 5xFAD transgenic mice. Mechanistically, our findings reveal that STING S-nitrosylation is critical in initiating signaling cascades by promoting the formation of disulfide-bonded STING oligomers. This leads to neuroinflammation early in the course of disease in vivo in 5xFAD mice with consequent synaptic loss. Collectively, our research supports the role of SNO-STING in neuroinflammation associated with AD, and points to a novel druggable cysteine residue in STING to prevent this S-nitrosylation reaction with its inherent inflammatory response. One Sentence SummaryS-Nitrosylation of STING triggers activation of cGAS-STING signaling in Alzheimers disease brain and subserves a novel link between excessive nitrosative stress and dysregulated innate immunity, thus contributing to disease progression.

neuroscience↗

bioRxiv2025

Virus glycoprotein nanodisc platform for vaccine design

Transmembrane glycoproteins of enveloped viruses are the targets of neutralizing antibodies and essential vaccine antigens. mRNA-LNP technology allows in situ production of transmembrane glycoproteins upon immunization, but biophysical characterization of transmembrane antigens and in vitro analysis of post-immunization antibody responses typically rely on soluble proteins. Here, we present a methodological platform for assembling transmembrane glycoprotein vaccine candidates into lipid nanodiscs. We demonstrate the utility of the nanodiscs in HIV membrane proximal external region (MPER)-targeting vaccine development by binding assays using surface plasmon resonance (SPR), ex vivo B cell sorting with fluorescence-activated cell sorting (FACS), and by determining the structure of a prototypical HIV MPER-targeting immunogen nanodisc in complex with three broadly neutralizing antibodies (bnAbs), including the MPER bnAb 10E8, to 3.5 [A] by cryogenic electron microscopy (cryo-EM), providing a template for structure-based immunogen design for MPER. Overall, the platform offers a tool for accelerating the development of next-generation viral vaccines.

immunology↗

bioRxiv2025

Proteomic profiling of primary cilia in the developing brain uncovers newregulators of cortical development

In the developing brain, the primary cilia of radial glial cells extend from the surface of the lateral ventricle, serving as the signaling hub to integrate environmental cues critical for brain formation. Dysfunctions in ciliary proteins contribute to a wide range of brain structural abnormalities in ciliopathies, yet identifying the bona fide ciliary protein components in the brain remains a significant challenge. Here, using proximity labeling and quantitative proteomics, we systematically charted proteins localized to the cilia of radial glial cells in the dorsal and ventral regions of the embryonic brain. From this dataset, we identified cohorts of new molecules intrinsic to the cilia at distinct regions of the developing brain. We validated the cilium localization of several components of the translation machinery and studied the mechanistic roles of ciliary candidates previously linked to brain malformation, including Marcks, a key regulator of radial glial polarity, and Ckap2l, a protein associated with Filippi syndrome. These results revealed previously unrecognized ciliary mechanisms that regulate brain development. Thus, our brain proteomic dataset provides a unique resource for understanding ciliary functions in brain development and the molecular etiology of developmental disorders.

developmental biology↗

bioRxiv2025

CRISPR-Cas9 Screening Reveals Microproteins Regulating Adipocyte Proliferation and Lipid Metabolism

Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation. SignificanceObesity and its associated metabolic disorders pose significant public health challenges, yet the molecular mechanisms regulating adipocyte function remain incompletely understood. Small open reading frames (smORFs) and their encoded microproteins represent an emerging class of regulatory elements with potential roles in metabolism. Here, we leveraged CRISPR/Cas9 screening to functionally characterize smORFs in adipocytes, identifying novel regulators of cell proliferation and lipid metabolism. Our findings demonstrate that conservation is not a prerequisite for smORF function, as we validated a mouse-specific microprotein that modulates adipocyte differentiation. This work establishes a robust pipeline for unbiased smORF discovery and highlights the potential for species-specific microproteins to regulate adipose biology. Future studies in human adipocytes may uncover additional microproteins with therapeutic relevance for obesity and metabolic disease.

cell biology↗

bioRxiv2025

Vaccination with mRNA-encoded membrane-bound HIV Envelope trimer induces neutralizing antibodies in animal models

A protective vaccine against HIV will likely need to induce broadly neutralizing antibodies (bnAbs) that engage relatively conserved epitopes on the HIV envelope glycoprotein (Env) trimer. Nearly all vaccine strategies to induce bnAbs require the use of relatively complex immunization regimens involving a series of different immunogens, most of which are Env trimers. Producing protein-based clinical material to evaluate such relatively complex regimens in humans presents major challenges in cost and time. Furthermore, immunization with HIV trimers as soluble proteins induces strong non-neutralizing responses to the trimer base, which is normally occluded on the virion. These base responses could potentially detract from the induction of nAbs and the eventual induction of bnAbs. mRNA vaccine platforms offer potential advantages over protein delivery for HIV vaccine development, including increased production speed, reduced cost, and the ability to deliver membrane-bound trimers that might facilitate improved immuno-focusing to non-base epitopes. We report the design of mRNA-delivered soluble and membrane-bound forms of a stabilized native-like Env trimer (BG505 MD39.3), initial immunogenicity evaluation in rabbits that triggered clinical evaluation, and more comprehensive evaluation of B cell, T cell, and antibody responses in non-human primates. mRNA-encoded membrane-bound Env immunization elicited reduced off-target base-directed Env responses and stronger neutralizing antibody responses, compared with mRNA-encoded soluble Env. Overall, mRNA delivery of membrane-bound Env appears promising for enhancing B cell responses to subdominant epitopes and facilitating rapid translation to clinical testing, which should assist HIV vaccine development. One Sentence SummaryHIV envelope trimer mRNA enables membrane-bound expression and represents a functional immunogen in pre-clinical mammalian models.

immunology↗

bioRxiv2024

Structural and Functional Insights into the Evolution of SARS-CoV-2 KP.3.1.1 Spike Protein

The JN.1-sublineage KP.3.1.1 recently emerged as the globally prevalent SARS-CoV-2 variant, demonstrating increased infectivity and antibody escape. We investigated how mutations and a deletion in the KP.3.1.1 spike protein (S) affect ACE2 binding and antibody escape. Mass spectrometry revealed a new glycan site at residue N30 and altered glycoforms at neighboring N61. Cryo-EM structures showed that the N30 glycan and rearrangement of adjacent residues did not significantly change the overall spike structure, up-down ratio of the receptor-binding domains (RBDs), or ACE2 binding. Furthermore, a KP.3.1.1 S structure with hACE2 further confirmed an epistatic effect between F456L and Q493E on ACE2 binding. Our analysis shows SARS-CoV-2 variants that emerged after late 2023 are now incorporating reversions to residues found in other sarbecoviruses, including the N30 glycan, Q493E, and others. Overall, these results inform on the structural and functional consequences of the KP.3.1.1 mutations, the current SARS-CoV-2 evolutionary trajectory, and immune evasion.

immunology↗

bioRxiv2024

Acetylation of lysine 82 initiates TDP-43 nuclear loss of function by disrupting its nuclear import

The hallmark of a spectrum of age-dependent neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), is a TDP-43 proteinopathy that includes nuclear loss of function and cytoplasmic aggregation. Here, reduced proteasome activity, as naturally occurs during aging, is shown to inhibit nuclear import of TDP-43. Quantitative mass spectrometry is used to determine that TDP-43 is the protein whose nuclear localization is most perturbed upon reduction in proteasome activity, culminating in elevated cytoplasmic TDP-43. Interaction of importin-1 with the bipartite classical nuclear localization sequence (cNLS) of TDP-43 is shown to be disrupted by partial proteasome inhibition but maintained by replacement with a PY-NLS that is recognized by importin-{beta}2. Mechanistically, this nuclear depletion of TDP-43 is shown to be driven by ubiquitination or acetylation of lysines 79, 82, and 84 within the cNLS when proteasome activity is reduced in human neurons. Specifically, acetylation at lysine 82 is sufficient to abolish TDP-43 binding to importin-1 and subsequent nuclear import of TDP-43. Moreover, using acetylation-specific TDP-43 antibodies, we detected acetylation of lysine 82 in the motor cortex of sporadic ALS patients but not control subjects. Our findings demonstrate that post-translational acetylation at lysine 82 of TDP-43 drives disruption of its importin-1-mediated nuclear import and is sufficient to initiate TDP-43 nuclear loss of function and cytoplasmic accumulation, evidence supporting acetylation as a plausible initiator of TDP-43 proteinopathies.

neuroscience↗

bioRxiv2024

Suppression of astrocyte BMP signaling improves fragile X syndrome molecular signatures and functional deficits

Fragile X syndrome (FXS) is a monogenic neurodevelopmental disorder with manifestations spanning molecular, neuroanatomical, and behavioral changes. Astrocytes contribute to FXS pathogenesis and show hundreds of dysregulated genes and proteins; targeting upstream pathways mediating astrocyte changes in FXS could therefore be a point of intervention. To address this, we focused on the bone morphogenetic protein (BMP) pathway, which is upregulated in FXS astrocytes. We generated a conditional KO (cKO) of Smad4 in astrocytes to suppress BMP signaling, and found this lessens audiogenic seizure severity in FXS mice. To ask how this occurs on a molecular level, we performed in vivo transcriptomic and proteomic profiling of cortical astrocytes, finding upregulation of metabolic pathways, and downregulation of secretory machinery and secreted proteins in FXS astrocytes, with these alterations no longer present when BMP signaling is suppressed. Functionally, astrocyte Smad4 cKO restores deficits in inhibitory synapses present in FXS auditory cortex. Thus, astrocytes contribute to FXS molecular and functional phenotypes, and targeting astrocytes can mitigate FXS symptoms.

neuroscience↗

bioRxiv2024

Diverse competitor B cell responses to a germline-targeting priming immunogen in human Ig loci transgenic mice

Broadly neutralizing antibodies (bnAbs) have promise to protect against HIV infection, but induction of bnAbs by immunization is an unsolved vaccine design challenge. Germline-targeting priming immunogens aim to initiate the induction of bnAbs by specifically activating rare bnAb-precursor B cells that can subsequently be matured using suitable heterologous boosting and shepherding immunogens. Several pre-clinical studies, and the IAVI G001 human clinical trial, have demonstrated the ability of a germline-targeting priming immunogen, eOD-GT8 60mer, to induce precursors of the VRC01 class of bnAbs. However, much less is known about B cells induced against other epitopes of the immunogen. Here, we performed unbiased analysis of B cells induced by eOD-GT8 60mers in Intelliselect Transgenic mice (Kymice) that are transgenic for the human Ig loci and produce human-like BCRs. B cells isolated with intact eOD-GT8 60mer nanoparticles showed a large diversity of non-VRC01-class B cells, with 38% unique clonotypes and only 5% of BCRs belonging to public lineages shared among all animals. We found that many competitors recognize epitopes in close proximity to or overlapping with the VRC01 epitope. These results indicate that optimal boosting of VRC01-class bnAb-precursor B cells primed by eOD-GT8 60mer might require a first-boost immunogen that minimizes recognition of competitor B cells, and such competitors isolated from Kymice could serve as valuable reagents for boost development.

immunology↗

bioRxiv2023

In vivo Protein Footprinting Reveals the Dynamic Conformational Changes of Proteome of Multiple Tissues in Progressing Alzheimer's Disease

Numerous studies have investigated changes in protein expression at the system level using proteomic mass spectrometry, but only recently have studies explored the structure of proteins at the proteome level. We developed covalent protein painting (CPP), a protein footprinting method that quantitatively labels exposed lysine, and have now extended the method to whole intact animals to measure surface accessibility as a surrogate of in vivo protein conformations. We investigated how protein structure and protein expression change as Alzheimers disease (AD) progresses by conducting in vivo whole animal labeling of AD mice. This allowed us to analyze broadly protein accessibility in various organs over the course of AD. We observed that structural changes of proteins related to energy generation, carbon metabolism, and metal ion homeostasis preceded expression changes in the brain. We found that proteins in certain pathways undergoing structural changes were significantly co-regulated in the brain, kidney, muscle, and spleen.

systems biology↗

bioRxiv2023

Evolving spike-protein N-glycosylation in SARS-CoV-2 variants

Since >3 years, SARS-CoV-2 has plunged humans into a colossal pandemic. Henceforth, multiple waves of infection have swept through the human population, led by variants that were able to partially evade acquired immunity. The co-evolution of SARS-CoV-2 variants with human immunity provides an excellent opportunity to study the interaction between viral pathogens and their human hosts. The heavily N-glycosylated spike-protein of SARS-CoV-2 plays a pivotal role in initiating infection and is the target for host immune-response, both of which are impacted by host-installed N-glycans. Using highly-sensitive DeGlyPHER approach, we compared the N-glycan landscape on spikes of the SARS-CoV-2 Wuhan-Hu-1 strain to seven WHO-defined variants of concern/interest, using recombinantly expressed, soluble spike-protein trimers, sharing same stabilizing-mutations. We found that N-glycan processing is conserved at most sites. However, in multiple variants, processing of N-glycans from high mannose- to complex-type is reduced at sites N165, N343 and N616, implicated in spike-protein function.

biochemistry↗

bioRxiv2022

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

microbiology↗

bioRxiv2021

A genome-edited β-cell model of Prader-Willi syndrome reveals chronic deficits in endoplasmic reticulum chaperones and insulin secretion

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in {beta}-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal (control) or active, paternal PWS-alleles. PWS {beta}-cells demonstrated a significant cell-autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, transcriptomic studies demonstrated that the broad reduction of ER chaperones originated from transcriptional downregulation without corresponding changes for Ins1 and Ins2. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS {beta}-cells. Consistent with reduced ER chaperones levels, PWS INS-1 {beta}-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. These findings suggest that a chronic shortage of ER chaperones in PWS {beta}-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic {beta}-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and {beta}-cell secretory pathway function.

genetics↗

bioRxiv2021

Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70s ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43s RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

cell biology↗

bioRxiv2021

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing via nuclear condensates

Cells respond to perturbations like inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Here, we found that depletion of arginine during inflammation decreased levels of a nuclear form of arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal protein and nuclear speckle component and that arginine depletion impacted both condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a novel mechanism whereby a tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

molecular biology↗

bioRxiv2021

An ultrasensitive method for analysis of viral spike N-glycoforms

Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.

biochemistry↗