Biology subjects

Elias, J. E.

Publications and source records attributed to Elias, J. E..

29 records · Page 2Linked to original sources

bioRxiv2020

Gut Microbiota-Targeted Diets Modulate Human Immune Status

Diet modulates the gut microbiome, and gut microbes, in turn, can impact the immune system. Here, we used two gut microbiota-targeted dietary interventions, plant-based fiber or fermented foods, to determine how each influences the human microbiome and immune system in healthy adults. Using a 17-week randomized, prospective study design combined with -omics measurements of microbiome and host, including extensive immune profiling, we found distinct effects of each diet. High-fiber consumers showed increased gut microbiome-encoded glycan-degrading CAZymes despite stable community diversity. Three distinct immunological trajectories in high fiber-consumers corresponded to baseline microbiota diversity. Alternatively, the high-fermented food diet steadily increased microbiota diversity and decreased inflammatory markers. The data highlight how coupling dietary interventions to deep and longitudinal immune and microbiome profiling can provide individualized and population-wide insight. Our results indicate that fermented foods may be valuable in countering the decreased microbiome diversity and increased inflammation pervasive in the industrialized society.

microbiology↗

bioRxiv2020

High-throughput cultivation of stable, diverse, fecal-derived microbial communities to model the intestinal microbiota

SummaryMechanistic understanding of the impacts of the gut microbiota on human health has been hampered by limited throughput in animal models. To enable systematic interrogation of gut-relevant microbial communities, here we generated hundreds of in vitro communities cultured from diverse stool samples in various media. Species composition revealed stool-derived communities that are phylogenetically complex, diverse, stable, and highly reproducible. Community membership depended on both medium and initial inoculum, with certain media preserving inoculum compositions. Different inocula yielded different community compositions, indicating their potential for personalized therapeutics. Communities were robust to freezing and large-volume culturing, enabling future translational applications. Defined communities were generated from isolates and reconstituted growth and composition similar to those of communities derived from stool inocula. Finally, in vitro experiments probing the response to ciprofloxacin successfully predicted many changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. Thus, stool-derived in vitro communities constitute a powerful resource for microbiota research.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2020

Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we find that NPC1s N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 and NPC1L1. The widely prescribed cholesterol uptake inhibitor, Ezetimibe, blocks NPC1L1; we show that interface residues that lie at the interface between NPC1L1s three extracellular domains comprise the drugs binding site. These data support a model in which cholesterol passes through the cores of NPC1/NPC1L1 proteins; concerted movement of various domains is needed for transfer and Ezetimibe blocks transport by binding to multiple domains simultaneously.

biochemistry↗

bioRxiv2020

High-Throughput Stool Metaproteomics: Method and Application to Human Specimens

Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, in comparison to competing technologies such as metagenomics and 16S rRNA sequencing, it is under-utilized due to its low throughput and the negative impact sample contaminants can have on highly sensitive mass spectrometry equipment. Here, we present a new stool proteomic processing pipeline that addresses these shortcomings in a highly reproducible and quantitative manner. Using this method, 290 samples from a dietary intervention study were processed in approximately 1.5 weeks, largely done by a single researcher. These data indicated a subtle but distinct monotonic increase in the number of significantly altered proteins between study participants on fiber- or fermented food-enriched diets. Lastly, we were able to classify study participants based on their diet-altered proteomic profiles, and demonstrated that classification accuracies of up to 89% could be achieved by increasing the number of subjects considered. Taken together, this study represents the first high throughout proteomic method for processing stool samples in a technically reproducible manner, and has the potential to elevate stool-based proteomics as an essential tool for profiling host-gut microbiome interactions in a clinical setting. ImportanceWidely available technologies based on DNA sequencing have been used to describe the kinds of microbes that might correlate with health and disease. However, mechanistic insight might be best achieved through careful study of the dynamic proteins at the interface between the foods we eat, our microbes, and ourselves. Mass-spectrometry-based proteomics has the potential to revolutionize our understanding of this complex system but its application to clinical studies has been hampered by low-throughput and laborious experimentation pipelines. In response, we developed SHT-Pro, the first high-throughput pipeline designed to rapidly handle large stool sample sets. With it, a single researcher can process over one hundred stool samples per week for mass spectrometry analysis, roughly 10 times faster than previous methods. Since SHT-Pro is fairly simple to implement using commercially available reagents, it should be easily adaptable to large-scale clinical studies.

systems biology↗

bioRxiv2019

Multiple layers of phospho-regulation coordinate metabolism and the cell cycle in budding yeast

The coordination of metabolism and growth with cell division is crucial for proliferation. While it has long been known that cell metabolism regulates the cell division cycle, it is becoming increasingly clear that the cell division cycle also regulates metabolism. In budding yeast, we previously showed that over half of all measured metabolites change concentration through the cell cycle indicating that metabolic fluxes are extensively regulated during cell cycle progression. However, how this regulation is achieved still remains poorly understood. Since both the cell cycle and metabolism are regulated to a large extent by protein phosphorylation, we here decided to measure the phosphoproteome through the budding yeast cell cycle. Specifically, we chose a cell cycle synchronisation strategy that avoids stress and nutrient-related perturbations of metabolism, and we grew the yeast on ethanol minimal medium to force cells to utilize their full biosynthetic repertoire. Using a tandem-mass-tagging approach, we found over 200 sites on metabolic enzymes and transporters to be phospho-regulated. These sites were distributed among many pathways including carbohydrate catabolism, lipid metabolism and amino acid synthesis and therefore likely contribute to changing metabolic fluxes through the cell cycle. Among all one thousand sites whose phosphorylation increases through the cell cycle, the CDK consensus motif and an arginine-directed motif were highly enriched. This arginine-directed R-R-x-S motif is associated with protein-kinase A, which regulates metabolism and promotes growth. Finally, we also found over one thousand sites that are dephosphorylated through the G1/S transition. We speculate that the phosphatase Glc7/ PP1, known to regulate both the cell cycle and carbon metabolism, may play an important role because its regulatory subunits are phospho-regulated in our data. In summary, our results identify extensive cell cycle dependent phosphorylation and dephosphorylation of metabolic enzymes and suggest multiple mechanisms through which the cell division cycle regulates metabolic signalling pathways to temporally coordinate biosynthesis with distinct phases of the cell division cycle.

cell biology↗

bioRxiv2019

Exercise conditioned plasma dampens inflammation via clusterin and boosts memory

Physical exercise seems universally beneficial to human and animal health, slowing cognitive aging and neurodegeneration. Cognitive benefits are tied to increased plasticity and reduced inflammation within the hippocampus, yet little is known about the factors and mechanisms mediating these effects. We discovered \"runner\" plasma, collected from voluntarily running mice, infused into sedentary mice recapitulates the cellular and functional benefits of exercise on the brain. Importantly, runner plasma reduces baseline neuroinflammatory gene expression and prominently suppresses experimentally induced brain inflammation. Plasma proteomic analysis shows a striking increase in complement cascade inhibitors including clusterin, which is necessary for the anti-inflammatory effects of runner plasma. Cognitively impaired patients participating in structured exercise for 6 months showed higher plasma clusterin levels, which correlated positively with improvements in endurance and aerobic capacity. These findings demonstrate the existence of anti-inflammatory \"exercise factors\" that are transferrable, benefit the brain, and are present in humans engaging in exercise.

neuroscience↗

bioRxiv2019

HIPK4 is essential for murine spermiogenesis

Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Ultrastructural analyses of HIPK4-null male germ cells reveal defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.

developmental biology↗

bioRxiv2018

Immuno-proteomic interrogation of dengue infection reveals novel HLA haplotype-specific MHC-I antigens

Broadly effective vaccines against dengue virus (DENV) infection have remained elusive, despite rising infection rates in the developing world. Infection-specific peptide ligands presented on Major Histocompatibility Complexes (MHC) open new avenues for developing T-cell-based interventions. Past efforts towards mapping viral MHC epitopes were based on computational predictions that only partially reflected actual antigen presentation. To empirically identify DENV-specific MHC ligands, we developed an immuno-proteomics approach for interrogating DENV- and self-derived MHC ligands from infected B-lymphocytes. Here, we report four fundamental findings: First, over 700 infection-specific MHC-ligands reflected host cellular responses to DENV that were not apparent from the proteome. Second, we report 121 viral MHC-I ligands (108 novel) which clustered into discrete hotspots across the DENV polyprotein, some of which spanned DENV polyprotein components, described here as MHC ligands for the first time. Third, we found DENV ligands which were distinctly presented by MHC alleles previously associated with either high or low anti-DENV response. Fourth, we demonstrate that while our in vitro assay only overlapped with a small fraction of previously described DENV T-cell epitopes, several novel MHC ligands identified here were recognized by T-cells from DENV-infected patients despite having low binding affinities. Together, these discoveries suggest that virus and host-derived MHC ligands have under-exploited potential for describing the cell biology of DENV infection, and as candidates for designing effective DENV vaccines.

immunology↗

bioRxiv2018

Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome and controls the abundance of key regulators of cellular function by selective protein degradation, but how foldingdefective proteins in the secretory system are selected from the large and diverse constellation of membrane and secretory proteins and efficiently delivered to proteasomes in the cytosol is not well understood. To determine the basis of substrate selectivity in human cells, we developed a transcriptional shut off approach to conduct parallel, unbiased, genome-wide CRISPR analysis of structurally and topologically diverse ER-associated degradation (ERAD) clients. Highly quantitative screen metrics allowed precise dissection of entire pathways, enabling identification of unique substrate-specific combinations of recognition and ubiquitin conjugation modules. Our analysis identified cytosolic ubiquitin conjugating machinery that has not been previously linked to ERAD but collaborates with membrane-integrated ubiquitin ligases to conjugate branched or mixed ubiquitin chains to promote efficient and processive substrate degradation.

cell biology↗

bioRxiv2018

Measuring proteomes with long strings: A new, unconstrained paradigm in mass spectrum interpretation

Thousands of protein post-translational modifications (PTMs) dynamically impact nearly all cellular functions. Mass spectrometry is well suited to PTM identification, but proteome-scale analyses are biased towards PTMs with existing enrichment methods. To measure the full landscape of PTM regulation, software must overcome two fundamental challenges: intractably large search spaces and difficulty distinguishing correct from incorrect identifications. Here, we describe TagGraph, software that overcomes both challenges with a string-based search method orders of magnitude faster than current approaches, and probabilistic validation model optimized for PTM assignments. When applied to a human proteome map, TagGraph tripled confident identifications while revealing thousands of modification types on nearly one million sites spanning the proteome. We expand known sites by orders of magnitude for highly abundant yet understudied PTMs such as proline hydroxylation, and derive tissue-specific insight into these PTMs roles. TagGraph expands our ability to survey the full landscape of PTM function and regulation.

bioinformatics↗

bioRxiv2018

The proteome of the malaria plastid organelle, a key anti-parasitic target

Malaria parasites (Plasmodium spp.) and related apicomplexan pathogens contain a non-photosynthetic plastid called the apicoplast. Derived from an unusual secondary eukaryote-eukaryote endosymbiosis, the apicoplast is a fascinating organelle whose function and biogenesis rely on a complex amalgamation of bacterial and algal pathways. Because these pathways are distinct from the human host, the apicoplast is an excellent source of novel antimalarial targets. Despite its biomedical importance and evolutionary significance, the absence of a reliable apicoplast proteome has limited most studies to the handful of pathways identified by homology to bacteria or primary chloroplasts, precluding our ability to study the most novel apicoplast pathways. Here we combine proximity biotinylation-based proteomics (BioID) and a new machine learning algorithm to generate a high-confidence apicoplast proteome consisting of 346 proteins. Critically, the high accuracy of this proteome significantly outperforms previous prediction-based methods and extends beyond other BioID studies of unique parasite compartments. Half of identified proteins have unknown function, and 77% are predicted to be important for normal blood-stage growth. We validate the apicoplast localization of a subset of novel proteins and show that an ATP-binding cassette protein ABCF1 is essential for blood-stage survival and plays a previously unknown role in apicoplast biogenesis. These findings indicate critical organellar functions for newly discovered apicoplast proteins. The apicoplast proteome will be an important resource for elucidating unique pathways derived from secondary endosymbiosis and prioritizing antimalarial drug targets.

microbiology↗