While it is generally recognized that misfolding of specific proteins can cause late-onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenorhabditis elegans. SDS-insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6–10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from E. coli. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation.

The transition of proteins targeted for autophagic degradation from the soluble to the insoluble phase is regulated in an ATG1-independent mechanism by TORC1. This process is likely a critical mechanism for maintaining protein homeostasis when challenged with proteomic stress.

It is well established that protein aggregation is associated with many neurodegenerative disorders including polyglutamine diseases, but a mechanistic understanding of the role of protein aggregates in the disease pathogenesis remains elusive. Previously thought to be the cause of cellular toxicity such as cellular dysfunction and cell death, protein aggregation is now proposed to serve a protective role by sequestering toxic oligomers from interfering with essential physiological processes. To investigate the relationship between protein aggregation and cellular toxicity, we have characterized and compared the effects of two GFP-fusion proteins that form aggregates in Saccharomyces cerevisiae, one with a polyasparagine repeat (GFP N104 ) and one without (GFP C ). Although both proteins can form microscopically visible GFP-positive aggregates, only the GFP N104 -containing aggregates exhibit morphological and biochemical characteristics that resemble the aggregates formed by mutant huntingtin in yeast cells. Formation of both the GFP C and GFP N104 aggregates depends on microtubules, while only the GFP N104 aggregate requires the chaperone Hsp104 and the prion Rnq1 and is resistant to SDS. Although no microscopically visible GFP N104 aggregates were observed in the hsp104D and rnq1D mutant cells, SDS-insoluble aggregates can still be detected by the filter trap assay. These observations argue that the GFP N104 -containing aggregates can exist in at least two distinct states in vivo. We also show that a nucleus-targeted GFP N104 interferes with transcription from two SAGA-dependant promoters and results in a decrease in cell viability. Overall, the results imply that the GFP N104 protein behaves similarly to the mutant huntingtin in yeast cells and provides a new model for investigating the interplay between protein aggregates and the associated phenotypes.

In recent decades, lakes have experienced unprecedented ice loss with widespread ramifications for winter ecological processes. The rapid loss of ice, resurgence of winter biology, and proliferation of remote sensing technologies, presents a unique opportunity to integrate disciplines to further understand the broad spatial and temporal patterns in ice loss and its consequences. Here, we summarize ice phenology records for 78 lakes in 12 countries across North America, Europe, and Asia to permit the inclusion and harmonization of in situ ice phenology observations in future interdisciplinary studies. These ice records represent some of the longest climate observations directly collected by people. We highlight the importance of applying the same definition of ice-on and ice-off within a lake across the time-series, regardless of how the ice is observed, to broaden our understanding of ice loss across vast spatial and temporal scales.

Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes.

A feature common to late onset proteinopathic disorders is an accumulation of toxic protein conformers and aggregates in affected tissues. In the search for potential drug targets, many studies used high-throughput screens to find genes that modify the cytotoxicity of misfolded proteins. A complement to this approach is to focus on strategies that use protein aggregation as a phenotypic readout to identify pathways that control aggregate formation and maintenance. Here we use natural variation between strains of budding yeast to genetically map loci that influence the aggregation of a polyglutamine-containing protein derived from a mutant form of huntingtin, the causative agent in Huntington disease. Linkage analysis of progeny derived from a cross between wild and laboratory yeast strains revealed two polymorphic loci that modify polyglutamine aggregation. One locus contains the gene RFU1 which modifies ubiquitination states of misfolded proteins targeted by the E3-ubiquitin ligase complex Rsp5. Activity of the Rsp5 complex, and the mammalian homolog NEDD4, are critical in maintaining protein homeostasis in response to proteomic stress. Our analysis also showed linkage of the aggregation phenotype to a distinct locus containing a gene encoding the Rsp5-interacting Bul2 protein. Allele-swap experiments validated the impact of both RFU1 and BUL2 on huntingtin aggregation. Furthermore, we found that the nematode Caenorhabditis elegans’ ortholog of Rsp5, wwp-1, also negatively regulates polyglutamine aggregation. Knockdown of the NEDD4 in human cells likewise altered polyglutamine aggregation. Taken together, these results implicate conserved processes involving the ubiquitin regulation network that modify protein aggregation and provide novel therapeutic targets for polyglutamine and other protein folding diseases.

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