Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by Proteasome Inhibition

Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity

Illuminating phylogeny and function of microbial dark matter in Sakinaw Lake

Microorganisms are the most abundant and diverse forms of life on Earth. Interconnected microbial communities drive matter and energy transformations integral to ecosystem functions and services through distributed metabolic networks innovated over 3.5 billion years of evolution. To effectively harness this metabolic potential it is necessary to chart uncultivated microbial community structure and function. Cultivation-independent studies indicate that over half of the microbial diversity on Earth belongs to uncultivated candidate divisions, also known as microbial dark matter (MDM). Here, I illuminate MDM structure and function in meromictic Sakinaw Lake on the Sunshine Coast of British Columbia Canada. Sakinaw Lake water column conditions quantified over 8 field campaigns were intimately associated with unprecedented abundance and diversity of MDM. Using network analysis and single cell genomics, co-occurrence patterns between MDM including OP9/JS-1, OP8 and methanogenic Archaea were linked with potential to perform syntrophic acetate oxidation, an important process in anaerobic digestion of organic matter in natural and engineered ecosystems. Single-cell and metagenome analysis revealed previously unrecognized nitrate reduction potential in candidate division OP3 and uncovered a novel archaeal lineage. In addition, numerous Fe-S oxidoreductases associated with MDM in Sakinaw Lake indicate the potential to couple sulfur oxidation to iron reduction. Taken together, my work establishes Sakinaw Lake as a natural laboratory in which to explore MDM structure and function, shines a spotlight on known and novel interactions and metabolic capabilities among these most enigmatic microorganisms, and points to potential biotechnological innovations based on cooperative interactions between MDM populations.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

From land to the Salish Sea – Towards an understanding of microplastics source, transport and fate

Contamination of marine ecosystems with microplastics (particle

A compendium of multi-omic sequence information from the Saanich Inlet water column.

Marine oxygen minimum zones (OMZs) are widespread regions of the ocean that are currently expanding due to global warming. While inhospitable to most metazoans, OMZs are hotspots for microbial mediated biogeochemical cycling of carbon, nitrogen and sulphur, contributing disproportionately to marine nitrogen loss and climate active trace gas production. Our current understanding of microbial community responses to OMZ expansion is limited by a lack of time-resolved data sets linking multi-omic sequence information (DNA, RNA, protein) to geochemical parameters and process rates. Here, we present six years of time-resolved multi-omic observations in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia, Canada that undergoes recurring changes in water column oxygenation status. This compendium provides a unique multi-omic framework for studying microbial community responses to ocean deoxygenation along defined geochemical gradients in OMZ waters

High-resolution phylogenetic microbial community profiling

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake's water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential

Saanich_TimeSeries_Historical_O2_DATA

Historical dissolved oxygen winkler measurements at Station S3 in Saanich Inlet from 1953 to 2007. This data includes, geographical coordinates, date (year and month), depth (meters), temperature (celcius degrees), salinity (PSU), density (sigma-theta), oxygen (milliliter per liter and micromolar). For detailed methods see the manuscript