A study of protein regulation by mass spectrometry-based methods

Understanding the function and regulation of proteins is critical to medical and physiological research. To achieve this, the modification status, as well as the interactions of proteins need to be defined. Mass spectrometry is often used to catalogue modifications but their effect on interactions is less often reported. In this thesis these two strands are combined to address a series of challenging complexes that underpin cellular processes from stress responses to energy production. Following an introduction to mass spectrometry and its applications to the study of protein interactions (Chapters 1 and 2), three protein assemblies are considered; Hsp70 chaperones, eukaryotic translation initiation factors, and F- and V-type ATP synthases. First, the oligomerisation of Hsp70 is studied (Chapter 3). Dimerisation is shown to be dependent on a highly-conserved phosphorylation site, suggesting a role for this modification in the regulation of protein complex assembly and targeting to antagonistic pathways. A study of eukaryotic translation initiation factor 2B (eIF2B) is presented (Chapter 4), with proteomics revealing clusters of modifications at protein interfaces. Chemical cross-linking is applied, eliminating proposed structural models and supporting a decameric assembly. A cognition-enhancing drug (known as ISRIB) is an inhibitor of the integrated stress response, and was found to stabilise the active decameric eIF2B assembly, rationalising the observed therapeutic effects of ISRIB. Finally, ATP synthases, membrane protein complexes which exhibit multiple forms of regulation, are investigated (Chapter 5). Positional information of small membrane subunits and the effect of post-translational modifications is deduced. The role of an endogenous inhibitor protein is investigated providing insights into its location, oligomeric state, and influence on nucleotide binding. Overall this thesis applies mass spectrometry to three critical protein assemblies and contributes a more complete understanding of their activity by uncovering aspects of their carefully-tuned regulatory mechanisms.</p

A study of protein regulation by mass spectrometry-based methods

Understanding the function and regulation of proteins is critical to medical and physiological research. To achieve this, the modification status, as well as the interactions of proteins need to be defined. Mass spectrometry is often used to catalogue modifications but their effect on interactions is less often reported. In this thesis these two strands are combined to address a series of challenging complexes that underpin cellular processes from stress responses to energy production. Following an introduction to mass spectrometry and its applications to the study of protein interactions (Chapters 1 and 2), three protein assemblies are considered; Hsp70 chaperones, eukaryotic translation initiation factors, and F- and V-type ATP synthases. First, the oligomerisation of Hsp70 is studied (Chapter 3). Dimerisation is shown to be dependent on a highly-conserved phosphorylation site, suggesting a role for this modification in the regulation of protein complex assembly and targeting to antagonistic pathways. A study of eukaryotic translation initiation factor 2B (eIF2B) is presented (Chapter 4), with proteomics revealing clusters of modifications at protein interfaces. Chemical cross-linking is applied, eliminating proposed structural models and supporting a decameric assembly. A cognition-enhancing drug (known as ISRIB) is an inhibitor of the integrated stress response, and was found to stabilise the active decameric eIF2B assembly, rationalising the observed therapeutic effects of ISRIB. Finally, ATP synthases, membrane protein complexes which exhibit multiple forms of regulation, are investigated (Chapter 5). Positional information of small membrane subunits and the effect of post-translational modifications is deduced. The role of an endogenous inhibitor protein is investigated providing insights into its location, oligomeric state, and influence on nucleotide binding. Overall this thesis applies mass spectrometry to three critical protein assemblies and contributes a more complete understanding of their activity by uncovering aspects of their carefully-tuned regulatory mechanisms.</p

Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90

Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90

Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the b-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F1FO adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid–bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome

Structure of the CRISPR Interference complex CSM reveals key similarities with Cascade

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I–III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family.Publisher PDFPeer reviewe