We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.
Proteomic
investigations of Alzheimer’s and Parkinson’s
disease have provided valuable insights into neurodegenerative disorders.
Thus far, these investigations have largely been restricted to bottom-up
approaches, hindering the degree to which one can characterize a protein’s
“intact” state. Top-down proteomics (TDP) overcomes
this limitation; however, it is typically limited to observing only
the most abundant proteoforms and of a relatively small size. Therefore,
fractionation techniques are commonly used to reduce sample complexity.
Here, we investigate gas-phase fractionation through high-field asymmetric
waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a
high complexity sample derived from Alzheimer’s disease (AD)
brain tissue, we describe how the addition of FAIMS to TDP can robustly
improve the depth of proteome coverage. For example, implementation
of FAIMS with external compensation voltage (CV) stepping at −50,
−40, and −30 CV could more than double the mean number
of non-redundant proteoforms, genes, and proteome sequence coverage
compared to without FAIMS. We also found that FAIMS can influence
the transmission of proteoforms and their charge envelopes based on
their size. Importantly, FAIMS enabled the identification of intact
amyloid beta (Aβ) proteoforms, including the aggregation-prone
Aβ1–42 variant which is strongly linked to
AD. Raw data and associated files have been deposited to the ProteomeXchange
Consortium via the MassIVE data repository with data set identifier
PXD023607.
Many AAA+ ATPases form hexamers that unfold protein substrates by translocating them through their central pore. Multiple structures have shown how a helical assembly of subunits binds a single strand of substrate, and indicate that translocation results from the ATP-driven movement of subunits from one end of the helical assembly to the other end. To understand how more complex substrates are bound and translocated, we demonstrated that linear and cyclic versions of peptides bind to the S. cerevisiae AAA+ ATPase Vps4 with similar affinities, and determined cryo-EM structures of cyclic peptide complexes. The peptides bind in a hairpin conformation, with one primary strand equivalent to the single chain peptide ligands, while the second strand returns through the translocation pore without making intimate contacts with Vps4. These observations indicate a general mechanism by which AAA+ ATPases may translocate a variety of substrates that include extended chains, hairpins, and crosslinked polypeptide chains.
Proper hematopoietic cell fate decisions require co-ordinated functions of transcription factors, their associated co-regulators, and histone-modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here, we describe a functional axis involving GFI1, SMYD2, and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -(8)KSKK(11)- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and co-ordinate control of hematopoietic cell fate in both normal and malignant settings.
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