Biology subjects

Topf, M.

Publications and source records attributed to Topf, M..

22 records · Page 2Linked to original sources

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↗

bioRxiv2018

Consensus clustering of herpesvirus protein interaction networks provides insights into their evolutionary relationship with bacteriophages

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the morbidity of some manifested symptoms but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterize herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, combining both experimentally supported and bioinformatic predictions. Our newly-developed consensus clustering approach allows us to extend the analysis beyond binary physical interactions and reveals higher order functional associations including that of pUS10 with capsid proteins. In-depth bioinformatics sequence analysis unraveled structural features of this 34-36 kDa protein reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are thought to share a common ancestry. This suggests that pUS10 could represent an evolutionary vestige between these two viral lineages. Using immunoaffinity purification-mass spectrometry we found that pUS10 specifically co-isolated with the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventual virion maturation. In summary, this study unveils new insights at both the system and molecular levels that can help better understand the complexity behind herpesvirus infections.

bioinformatics↗

bioRxiv2018

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

The membrane attack complex (MAC) is one of the immune systems first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant {beta}-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how {beta}-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

biophysics↗

bioRxiv2018

Evolution of the CLTCL1 Gene Encoding CHC22 Clathrin Reveals Selection Influencing CHC22’s Role in Human Glucose Metabolism

CHC22 clathrin plays a key role in intracellular membrane traffic of the insulin-responsive glucose transporter GLUT4 in humans. We performed population genetic and phylogenetic analyses of the CHC22-encoding CLTCL1 gene, revealing independent gene loss in at least two vertebrate lineages, after arising from gene duplication. All vertebrates retained the paralogous CLTC gene encoding CHC17 clathrin, which mediates endocytosis. For vertebrates retaining CLTCL1, strong evidence for purifying selection supports CHC22 functionality. All human populations maintained two high frequency CLTCL1 allelic variants, encoding either methionine or valine at position 1316. Functional studies indicated that CHC22-V1316, which is more frequent in farming populations than in hunter-gatherers, has different cellular dynamics than M1316-CHC22 and is less effective at controlling GLUT4 membrane traffic, attenuating its insulin-regulated response. These analyses suggest that ancestral human dietary change influenced selection of allotypes that affect CHC22s role in metabolism and have potential to differentially influence the human insulin response.

genetics↗