Highlights d Strategy for affinity purification of native chromatin fragments d Heterochromatin and euchromatin have overlapping and non-overlapping proteomes d The heterochromatin proteome includes inner nuclear membrane proteins and nucleoporins d Inner-ring nucleoporins are required for clustering and maintenance of heterochromatin
The proteome is the functional entity of the cell, and perturbations of a cellular system almost always cause changes in the proteome. These changes are a molecular fingerprint, allowing characterization and a greater understanding of the effect of the perturbation on the cell as a whole. Monitoring these changes has therefore given great insight into cellular responses to stress and disease states, and analytical platforms to comprehensively analyze the proteome are thus extremely important tools in biological research. Mass spectrometry has evolved as the most relevant technology to characterize proteomes in a comprehensive way. However, due to a lack of throughput capacity of mass spectrometry-based proteomics, researchers frequently use measurement of mRNA levels to approximate proteome changes. Growing evidence of substantial differences between mRNA and protein levels as well as recent improvements in mass spectrometry-based proteomics are heralding an increased use of mass spectrometry for comprehensive proteome mapping. Here we describe the use of multiplexed quantitative proteomics using isobaric labeling with tandem mass tags (TMT) for the simultaneous quantitative analysis of five cancer cell proteomes in biological duplicates in one mass spectrometry experiment.
SUMMARY
PUMA is a pro-apoptotic BCL-2 family member that drives the apoptotic response to a diversity of p53-dependent and independent cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent pro-apoptotic properties remains a high priority goal. Here, we report the synthesis of PUMA SAHBs, structurally-stabilized PUMA BH3 helices that, in addition to broadly targeting anti-apoptotic proteins, directly bind to BAX. NMR, photocrosslinking, and biochemical analyses revealed that PUMA SAHBs engage an α1/α6 trigger site on BAX to initiate its functional activation. We further demonstrated that a cell-permeable PUMA SAHB analog induces apoptosis in neuroblastoma cells and, like expressed PUMA protein, engages BCL-2, MCL-1 and BAX. Thus, we find that PUMA BH3 is a dual anti-apoptotic inhibitor and pro-apoptotic direct activator, and its mimetics may serve as effective pharmacologic triggers of apoptosis in resistant human cancers.
Hydrocarbon stapling has been applied to restore and stabilize the α-helical structure of bioactive peptides for biochemical, structural, cellular, and in vivo studies. The peptide sequence, in addition to the composition and location of the installed staple, can dramatically influence the properties of stapled peptides. As a result, constructs that appear similar can have distinct functions and utilities. Here, we perform a side-by-side comparison of stapled peptides modeled after the pro-apoptotic BIM BH3 helix to highlight these principles. We confirm that replacing a salt-bridge with an i, i + 4 hydrocarbon staple does not impair target binding affinity and instead can yield a biologically and pharmacologically enhanced α-helical peptide ligand. Importantly, we demonstrate by electron microscopy that the pro-apoptotic activity of a stapled BIM BH3 helix correlates with its capacity to achieve cellular uptake without membrane disruption and accumulate at the organellar site of mechanistic activity.
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