RESUMO
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. It stipulates that only proteins for which at least one monolink or intra-protein cross-link has been identified within a given data set are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data.
Assuntos
Proteoma , Espectrometria de Massas , Proteoma/química , Reagentes de Ligações Cruzadas/químicaRESUMO
Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif of FUS as a key driver of condensate aging. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.
Assuntos
Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , RNA/metabolismo , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Small heat-shock proteins (sHSPs) are important members of the cellular stress response in all species. Their best-described function is the binding of early unfolding states and the resulting prevention of protein aggregation. Many sHSPs exist as a polydisperse composition of oligomers, which undergoes changes in subunit composition, folding status, and relative distribution upon heat activation. To date, only an incomplete picture of the mechanism of sHSP activation exists; in particular, the molecular basis of how sHSPs bind client proteins and mediate client specificity is not fully understood. In this study, we have applied cross-linking mass spectrometry (XL-MS) to obtain detailed structural information on sHSP activation and client binding for yeast Hsp26. Our cross-linking data reveals the middle domain of Hsp26 as a client-independent interface in multiple Hsp26::client complexes and indicates that client specificity is likely mediated via additional binding sites within its α-crystallin domain and C-terminal extension. Our quantitative XL-MS data underpins the middle domain as the main driver of heat-induced activation and client binding but shows that global rearrangements spanning all domains of Hsp26 take place simultaneously. We also investigated a Hsp26::client complex in the presence of Ssa1 (Hsp70) and Ydj1(Hsp40) at the initial stage of refolding and observe that the interaction between refolding chaperones is altered by the presence of a client protein, pointing to a mechanism where the interaction of Ydj1 with the HSP::client complex initiates the assembly of the active refolding machinery.
Assuntos
Proteínas de Choque Térmico , Espectrometria de Massas , Proteínas de Choque Térmico/química , Humanos , Estrutura Quaternária de ProteínaRESUMO
Proteome-wide cross-linking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In this study we demonstrate both experimentally and by a kinetic model that this bias is also caused by the propensity of cross-links to preferentially form on high abundant proteins and not by the inability to detect cross-links due to limitations in current technology. We further show, by using both an in vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that parameters optimized toward a pseudo first order kinetics model result in a significant increase in the detection of lower-abundant proteins on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide cross-linking studies and demonstrates how to address a larger part of the proteome by cross-linking.