Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.

Aggregation-prone proteins (APPs) have been implicated in numerous human diseases but the underlying mechanisms are incompletely understood. Here we comparatively analysed cellular responses to different APPs. Our study is based on a systematic proteomic and phosphoproteomic analysis of a set of yeast proteotoxicity models expressing different human disease-related APPs, which accumulate intracellular APP inclusions and exhibit impaired growth. Clustering and functional enrichment analyses of quantitative proteome-level data reveal that the cellular response to APP expression, including the chaperone response, is specific to the APP, and largely differs from the response to a more generalized proteotoxic insult such as heat shock. We further observe an intriguing association between the subcellular location of inclusions and the location of the cellular response, and provide a rich dataset for future mechanistic studies. Our data suggest that care should be taken when designing research models to study intracellular aggregation, since the cellular response depends markedly on the specific APP and the location of inclusions. Further, therapeutic approaches aimed at boosting protein quality control in protein aggregation diseases should be tailored to the subcellular location affected by inclusion formation. SIGNIFICANCE: We have examined the global cellular response, in terms of protein abundance and phosphorylation changes, to the expression of five human neurodegeneration-associated, aggregation-prone proteins (APPs) in a set of isogenic yeast models. Our results show that the cellular response to each APP is unique to that protein, is different from the response to thermal stress, and is associated with processes at the subcellular location of APP inclusion formation. These results further our understanding of how cells, in a model organism, respond to expression of APPs implicated in neurodegenerative diseases like Parkinson's, Alzheimer's, and ALS. They have implications for mechanisms of toxicity as well as of protective responses in the cell. The specificity of the response to each APP means that research models of these diseases should be tailored to the APP in question. The subcellular localization of the response suggest that therapeutic interventions should also be targeted within the cell.

Parkinson’s disease (PD), Alzheimer’s disease (AD) and Amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases hallmarked by the formation of toxic protein aggregates. However, targeting these aggregates therapeutically have thus far shown no success. The treatment of AD has remained particularly problematic since no new drugs have been approved in the last 15 years. Therefore, novel therapeutic targets need to be identified and explored. Here, through the integration of genomic and proteomic data, a set of proteins with strong links to α-synuclein-aggregating neurodegenerative diseases was identified. We propose 17 protein targets that are likely implicated in neurodegeneration and could serve as potential targets. The human phosphatidylinositol 5-phosphatase synaptojanin-1, which has already been independently confirmed to be implicated in Parkinson’s and Alzheimer’s disease, was among those identified. Despite its involvement in PD and AD, structural aspects are currently missing at the molecular level. We present the first atomistic model of the 5-phosphatase domain of synaptojanin-1 and its binding to its substrate phosphatidylinositol 4,5-bisphosphate (PIP2). We determine structural information on the active site including membrane-embedded molecular dynamics simulations. Deficiency of charge within the active site of the protein is observed, which suggests that a second divalent cation is required to complete dephosphorylation of the substrate. The findings in this work shed light on the protein’s binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and give additional insight for future targeting of the protein active site, which might be of interest in neurodegenerative diseases where synaptojanin-1 is overexpressed.

The Biology/Disease-driven (B/D) working groups of the Human Proteome Project are alliances of research groups aimed at developing or improving proteomic tools to support specific biological or disease-related research areas. Here, we describe the activities and progress to date of the B/D working group focused on protein aggregation diseases (PADs). PADs are characterized by the intra- or extracellular accumulation of aggregated proteins and include devastating diseases such as Parkinson's and Alzheimer's disease and systemic amyloidosis. The PAD B/D working group aims for the development of proteomic assays for the quantification of aggregation-prone proteins involved in PADs to support basic and clinical research on PADs. Because the proteins in PADs undergo aberrant conformational changes, a goal is to quantitatively resolve altered protein structures and aggregation states in complex biological specimens. We have developed protein-extraction protocols and a set of mass spectrometric (MS) methods that enable the detection and quantification of proteins involved in the systemic and localized amyloidosis and the probing of aberrant protein conformational transitions in cell and tissue extracts. In several studies, we have demonstrated the potential of MS-based proteomics approaches for specific and sensitive clinical diagnoses and for the subtyping of PADs. The developed methods have been detailed in both protocol papers and manuscripts describing applications to facilitate implementation by nonspecialized laboratories, and assay coordinates are shared through public repositories and databases. Clinicians actively involved in the PAD working group support the transfer to clinical practice of the developed methods, such as assays to quantify specific disease-related proteins and their fragments in biofluids and multiplexed MS-based methods for the diagnosis and typing of systemic amyloidosis. We believe that the increasing availability of tools to precisely measure proteins involved in PADs will positively impact research on the molecular bases of these diseases and support early disease diagnosis and a more-confident subtyping.

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101(Mms22) ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101(Mms22) E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.

Although chromosome partitioning during mitosis is well studied, the molecular mechanisms that allow proper segregation of cytoplasmic organelles in human cells are poorly understood. Here we show that mitochondria interact with growing microtubule tips and are transported towards the daughter cell periphery at the end of mitosis. This phenomenon is promoted by the direct and cell cycle-dependent interaction of the mitochondrial protein Miro and the cytoskeletal-associated protein Cenp-F. Cenp-F is recruited to mitochondria by Miro at the time of cytokinesis and associates with microtubule growing tips. Cells devoid of Cenp-F or Miro show decreased spreading of the mitochondrial network as well as cytokinesis-specific defects in mitochondrial transport towards the cell periphery. Thus, Miro and Cenp-F promote anterograde mitochondrial movement and proper mitochondrial distribution in daughter cells.

We describe a proteomic screening approach based on the concept of sentinel proteins, biological markers whose change in abundance characterizes the activation state of a given cellular process. Our sentinel assay simultaneously probed 188 biological processes in Saccharomyces cerevisiae exposed to a set of environmental perturbations. The approach can be applied to analyze responses to large sets of uncharacterized perturbations in high throughput.

Coupling limited proteolysis and a proteomics workflow enables measurement of both subtle and wholesale protein conformational changes in a eukaryotic proteome.

Construction and intracellular targeting of eukaryotic pre‐ribosomal particles involve a multitude of diverse transiently associating trans‐acting assembly factors, energy‐consuming enzymes, and transport factors. The ability to rapidly and reliably measure co‐enrichment of multiple factors with maturing pre‐ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise contribution of >50 energy‐consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity‐capture, and selected reaction monitoring mass spectrometry (SRM‐MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre‐60S particles after nuclear export. We uncovered assembly factors that travel with pre‐60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre‐60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport. A combination of genetic trapping, affinity‐capture and selected reaction monitoring mass spectrometry is used to characterize the dynamic proteome of pre‐60S ribosomal particles after nuclear export. These results identify Bud20 as a novel shuttling factor for pre‐60S export. A combination of genetic trapping, affinity‐capture and selected reaction monitoring mass spectrometry is used to characterize the dynamic proteome of pre‐60S ribosomal particles after nuclear export. These results identify Bud20 as a novel shuttling factor for pre‐60S export.