Biology subjects

Budnik, B.

Publications and source records attributed to Budnik, B..

32 records · Page 2Linked to original sources

bioRxiv2022

A Proximity Biotinylation Assay with a Host Protein Bait Reveals Multiple Factors Modulating Enterovirus Replication

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, arguing that the GBF1 environment is linked to the viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified those previously associated with the enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins many of which normally localize in the nucleus constituted the largest group, underscoring the massive release of nuclear factors in the cytoplasm of infected cells and their involvement in the viral replication. Analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly increase the knowledge about the organization of enterovirus replication organelles and may provide new targets for anti-viral interventions.

microbiology↗

bioRxiv2022

Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins

Non-native conformations drive protein misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well-suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

biophysics↗

bioRxiv2022

Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality

Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions led to two outcomes -- removal of cytoplasmic material and stem cell division, or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

developmental biology↗

bioRxiv2021

Formation and recycling of an active epigenetic mark mediated by cell cycle-specific RNAs

The mechanisms by which epigenetic modifications are established in gene regulatory regions of active genes remain poorly understood. The data presented show that the establishment and recycling of a major epigenetic mark, the acetylated form of the replacement histone H2A.Z, is regulated by cell cycle-specific long noncoding RNAs encoded in regions adjacent to the promoters of active genes. These transcripts, termed SPEARs (S Phase EArly RNAs), are induced in early S phase: their expression precedes that of the downstream genes on which they exert their regulatory action. SPEARs drive the modification and deposition of the acetylated form of histone H2A.Z by bringing together the replacement histone and the histone acetyl transferase TIP60. This widespread bimodal pathway constitutes a novel RNA-mediated mechanism for the establishment of epigenetic marks and cell-specific epigenetic profiles, thereby providing a unifying explanation for the accuracy and persistence of epigenetic marks on chromatin.

genetics↗

bioRxiv2021

Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

neuroscience↗

bioRxiv2021

Development of photo-lenalidomide for cellular target identification

The thalidomide analog lenalidomide is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photo-lenalidomide, a lenalidomide probe with a photo-affinity label and enrichment handle, for target identification by chemical proteomics. After evaluating a series of lenalidomide analogs, we identified a specific amide linkage to lenalidomide that allowed for installation of the desired functionality, while preserving the substrate degradation profile, phenotypic anti-proliferative and immunomodulatory properties of lenalidomide. Photo-lenalidomide maintains these properties by enhancing binding interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using photo-lenalidomide, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells, and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by photolenalidomide and forms a complex with CRBN in the presence of lenalidomide, but is itself not ubiquitylated or degraded. These data point to the potentially broader array of substrates induced by ligands to CRBN that may or may not be degraded, which can be revealed by the highly translatable application of photo-lenalidomide and chemical proteomics in additional biological settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/452075v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1619364org.highwire.dtl.DTLVardef@113a07corg.highwire.dtl.DTLVardef@18d36acorg.highwire.dtl.DTLVardef@8e64_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

In-depth analysis of proteomic and genomic fluctuations during the time course of human embryonic stem cells directed differentiation into beta cells.

Pluripotent stem cells (PSC) endocrine differentiation at a large scale allows sampling of transcriptome and proteome with phosphoproteome (proteoform) at specific time points. We describe the dynamic time course of changes in cells undergoing directed beta-cell differentiation and show target proteins or previously unknown phosphorylation of critical proteins in pancreas development, NKX6-1, and Chromogranin A (CHGA). We describe fluctuations in the correlation between gene expression, protein abundance, and phosphorylation, which follow differentiation protocol perturbations of cell fates at all stages to identify proteoform profiles. Our computational modeling recognizes outliers on a phenomic landscape of endocrine differentiation, and we outline several new biological pathways involved. We have validated our proteomic data by analyzing two independent single-cell RNA sequencing datasets for in-vitro pancreatic islet productions using the same cell starting material and differentiation protocol and corroborating our findings for several proteins suggest as targets for future research. Moreover, our single-cell analysis combined with proteoform data places new protein targets within the specific time point and at the specific pancreatic lineage of differentiating stem cells. We also suggest that non-correlating proteins abundances or new phosphorylation motifs of NKX6.1 and CHGA point to new signaling pathways that may play an essential role in beta-cell development. We present our findings for the research communitys use to improve endocrine differentiation protocols and developmental studies.

cell biology↗

bioRxiv2018

6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion

The proteinaceous extracellular matrix (ECM) is vital for cancer cell survival, proliferation, migration, and differentiation. However, little is known regarding metabolic pathways required in the ECM secretion process. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for endoplasmic reticulum (ER) structural integrity and protein secretion. Chemical inhibition or genetic suppression of its activity led to cell stress accompanied by significantly expanded ER volume and can be rescued by compensating glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD exemplifies a nexus of cytosolic carbohydrate metabolism and protein secretion.

biochemistry↗

bioRxiv2018

Automated sample preparation for high-throughput single-cell proteomics

A major limitation to applying quantitative LC-MS/MS proteomics to small samples, such as single cells, are the losses incured during sample cleanup. To relieve this limitation, we developed a Minimal ProteOmic sample Preparation (mPOP) method for culture-grown mammalian cells. mPOP obviates cleanup and thus eliminates cleanup-related losses while expediting sample preparation and simplifying its automation. Bulk SILAC samples processed by mPOP or by conventional urea-based methods indicated that mPOP results in complete cell lysis and accurate relative quantification. We integrated mPOP lysis with the Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) sample preparation, and benchmarked the quantification of such samples on a Q-exactive instrument. The results demonstrate low noise and high technical reproducibility. Then, we FACS sorted single U-937, HEK-293, and mouse ES cells into 96-well plates and analyzed them by automated mPOP and SCoPE-MS. The quantified proteins enabled separating the single cells by cell-type and cell-division-cycle phase.

systems biology↗

bioRxiv2018

Cohesin interacts with a panoply of splicing factors required for cell cycle progression and genomic organization

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Here we report that endogenous human cohesin interacts with a panoply of splicing factors and RNA binding proteins, including diverse components of the U4/U6.U5 tri-snRNP complex and several splicing factors that are commonly mutated in cancer. The interactions are enhanced during mitosis, and the interacting splicing factors and RNA binding proteins follow the cohesin cycle and prophase pathway of regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors results in stereotyped cell cycle arrests and alterations in genomic organization. These data support the hypothesis that splicing factors and RNA binding proteins control cell cycle progression and genomic organization via regulated interactions with cohesin and chromatin.\n\nOne Sentence SummaryEndogenous tagging reveals that cohesin interacts with diverse chromatin-bound splicing factors that regulate cell cycle progression and genomic organization in human cells.

cell biology↗

bioRxiv2018

Catalysis of cataract-associated human γD crystallin aggregation via dynamic disulfide exchange

Several mutations in human {gamma}D-crystallin (H{gamma}D), a long-lived eye lens protein, cause misfolding and aggregation, leading to cataract. Surprisingly, wild-type H{gamma}D catalyzes aggregation of its cataract related W42Q variant while itself remaining soluble - the inverse of the classical prion-like scenario whereby misfolded polypeptides catalyze aggregation of natively folded ones. The search for a biochemical mechanism of catalysis of W42Q aggregation by WT has revealed that WT H{gamma}D can transfer a disulfide bond to the W42Q variant. The transferred disulfide kinetically traps an aggregation-prone intermediate made accessible by the W42Q mutation, facilitating light-scattering aggregation of the W42Q variant. The aggregating variant thus becomes a disulfide sink, removing the disulfides from solution. Such redox \"hot potato\" competitions among wild-type and mutant or modified polypeptides may be relevant for many long-lived proteins that function in oxidizing environments. In these cases aggregation may be forestalled by inhibiting disulfide flow toward damaged polypeptides.

biochemistry↗

bioRxiv2017

Quantifying homologous proteins and proteoforms

Many proteoforms - arising from alternative splicing, post-translational modifications (PTMs), or paralogous genes - have distinct biological functions, such as histone PTM proteoforms. However, their quantification by existing bottom-up mass-spectrometry (MS) methods is undermined by peptide-specific biases. To avoid these biases, we developed and implemented a first-principles model (HIquant) for quantifying proteoform stoichiometries. We characterized when MS data allow inferring proteoform stoichiometries by HIquant, derived an algorithm for optimal inference, and demonstrated experimentally high accuracy in quantifying fractional PTM occupancy without using external standards, even in the challenging case of the histone modification code. A HIquant server is implemented at: https://web.northeastern.edu/slavov/2014_HIquant/

systems biology↗

bioRxiv2017

Pervasive Discordance Between mRNA And Protein Expression During Embryonic Stem Cell Differentiation

During in vitro differentiation, pluripotent stem cells undergo extensive remodeling of their gene expression profile. While studied extensively at the transcriptome level, much less is known about protein dynamics. Here, we measured mRNA and protein levels of 7459 genes during differentiation of embryonic stem cells (ESCs). This comprehensive data set revealed pervasive discordance between mRNA and protein. The high temporal resolution of the data made it possible to determine protein turnover rates genome-wide by fitting a kinetic model. This model further enabled us to systematically identify dynamic post-transcriptional regulation. Moreover, we linked different modes of regulation to the function of specific gene sets. Finally, we showed that the kinetic model can be applied to singlecell transcriptomics data to predict protein levels in differentiated cell types. In conclusion, our comprehensive data set, easily accessible through a web application, is a valuable resource for the discovery of post-transcriptional regulation in ESC differentiation.

systems biology↗

bioRxiv2017

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

systems biology↗