Mono- and Intralink Filter (Mi-Filter) To Reduce False Identifications in Cross-Linking Mass Spectrometry Data.

Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow.

Allosteric control of Ubp6 and the proteasome via a bidirectional switch.

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect.

Molecular and cellular dynamics of the 26S proteasome.

In eukaryotic cells, the ubiquitin-proteasome system serves to remove proteins that are either dysfunctional or no longer needed. The 26S proteasome is a 2.5 MDa multisubunit complex comprising the 20S core particle, where degradation is executed, and one or two regulatory particles which prepare substrates for degradation.

An integrated workflow for crosslinking mass spectrometry.

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features.

Expanded Coverage of the 26S Proteasome Conformational Landscape Reveals Mechanisms of Peptidase Gating.

The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo-electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics.

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation.

Macrophages not only produce multiple cytokines but also respond to multiple cytokines, which likely shapes the ultimate response of the population. To determine the role of paracrine signaling in shaping the profile of inflammatory cytokines secreted by macrophages in response to stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS), we combined multiplexed, microwell-based measurements of cytokine secretion by single cells with analysis of cytokine secretion by cell populations. Loss of paracrine signaling as a result of cell isolation reduced the secretion by macrophage-like U937 cells and human monocyte-derived macrophages (MDMs) of a subset of LPS-stimulated cytokines, including interleukin-6 (IL-6) and IL-10.

Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands.

Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation.

Regulation of Torsin ATPases by LAP1 and LULL1.

TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1.