Early butyrate induced acetylation of histone H4 is proteoform specific and linked to methylation state

<p>Histone posttranslational modifications (PTMs) help regulate DNA templated processes; however, relatively little work has unbiasedly explored the single-molecule combinations of histone PTMs, their dynamics on short timescales, or how these preexisting histone PTMs modulate further histone modifying enzyme activity. We use quantitative top down proteomics to unbiasedly measure histone H4 proteoforms (single-molecule combinations of PTMs) upon butyrate treatment. Our results show that histone proteoforms change in cells within 10 minutes of application of sodium butyrate. Cells recover from treatment within 30 minutes after removal of butyrate. Surprisingly, K20me2 containing proteoforms are the near-exclusive substrate of histone acetyltransferases upon butyrate treatment. Single-molecule hierarchies of progressive PTMs mostly dictate the addition and removal of histone PTMs (K16ac > K12ac ≥ K8ac > K5ac, and the reverse on recovery). This reveals the underlying single-molecule mechanism that explains the previously reported but indistinct and unexplained patterns of H4 acetylation. Thus, preexisting histone PTMs strongly modulate histone modifying enzyme activity and this suggests that proteoform constrained reaction pathways are crucial mechanisms that enable the long-term stability of the cellular epigenetic state.</p

Mapping the contact surfaces in the Lamin A:AIMP3 complex by hydrogen/deuterium exchange FT-ICR mass spectrometry

<div><p>Aminoacyl-tRNA synthetases-interacting multifunctional protein3 (AIMP3/p18) is involved in the macromolecular tRNA synthetase complex via its interaction with several aminoacyl-tRNA synthetases. Recent reports reveal a novel function of AIMP3 as a tumor suppressor by accelerating cellular senescence and causing defects in nuclear morphology. AIMP3 specifically mediates degradation of mature Lamin A (LmnA), a major component of the nuclear envelope matrix; however, the mechanism of how AIMP3 interacts with LmnA is unclear. Here we report solution-phase hydrogen/deuterium exchange (HDX) for AIMP3, LmnA, and AIMP3 in association with the LmnA C-terminus. Reversed-phase LC coupled with LTQ 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) results in high mass accuracy and resolving power for comparing the D-uptake profiles for AIMP3, LmnA, and their complex. The results show that the AIMP3-LmnA interaction involves one of the two putative binding sites and an adjacent novel interface on AIMP3. LmnA binds AIMP3 via its extreme C-terminus. Together these findings provide a structural insight for understanding the interaction between AIMP3 and LmnA in AIMP3 degradation.</p></div

Sequence coverage for proteolytic peptides (5–30 aa in length) common to free His-Tev-AIMP3 and His-Tev-AIMP3 in complex (Top); and free His-Strep-TrxA-LmnA and His-Strep-TrxA-LmnA in complex (bottom).

<p>Peptides containing less than 5 or more than 30 amino acids are not considered, due to increased ambiguity and poor sequence localization. The displayed segments cover 100% of the sequences based on the common segments.</p

H/D exchange results for free and complexed AIMP3 with LmnA.

<p>For each of the proteolytic peptides common to free and bound AIMP3, the relative D-uptake change for AIMP3 on binding to LmnA (ARDD) is calculated as described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181869#pone.0181869.e001" target="_blank">Eq 1</a>. Peptide regions with significant deuterium uptake differences are mapped onto the crystal structure. Top: AIMP3 shows decreases in D-uptake for segments 91–96 and 134–152 upon binding to LmnA, consistent with the binding interface between AIMP3 and LmnA. Bottom: ARDD mapped onto the crystal structure (PDB 2UZ8). Note that segments 91–96 and 134–139 (red circle) are spatially close to each other.</p

Deuterium uptake profiles (data points) and maximum-entropy fits (smooth curves [54]) vs. H/D exchange period (log₁₀ scale) for selected segments of free and complexed AIMP3.

<p>Segment 91–96 of putative binding Interface I exhibits a significant decrease in D-uptake upon forming the complex. Segments 127–133 and 158–171 constitute putative Interface II, but show no change in D-uptake. Significant decreases are also observed for segments 134–139 and 143–152, thereby defining the AIMP3 binding surface to LmnA.</p

H/D exchange results for free and complexed LmnA with AIMP3.

<p>ARDD for LmnA is calculated as for AIMP3. Top: LmnA shows decreased D-uptake for segment 203–209 (641–647) upon binding AIMP3. Bottom: Deuterium uptake profiles (data points) and maximum-entropy fits (smooth curves [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181869#pone.0181869.ref054" target="_blank">54</a>]) vs. H/D exchange period (log₁₀ scale) for selected segments of free and complexed LmnA. Left: Selected peptide 64–73, representing peptides in the His-Strep-TrxA tag, shows unaltered deuteration level. Right: Peptide 203–209 (641–647) shows significantly decreased D-uptake upon binding AIMP3.</p

HDX heat map for deuterium uptake by free His-Tev-AIMP3.

<p>The His-Tev tag sequence is in grey. The deuteration level percentage is calculated by dividing the observed deuterium uptake by the total number of amide hydrogens (not counting proline) in that segment. For each peptide, the calculated deuteration level for each HDX incubation period (top left, proceeding from top to bottom: incubation periods of 0.5, 1, 2, 4, 8, 15, 30, 60, 120, and 240 min) is mapped onto the sequence. Secondary structure is noted on top of the sequence (PDB 2UZ8) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181869#pone.0181869.ref053" target="_blank">53</a>]. Alpha helices and beta strands are numbered in order from N to C terminus.</p

HDX heat map for deuterium uptake by free His-Strep-TrxA-LmnA.

<p>The His-Strep-TrxA tag sequence is in gray. The deuteration level percentage is calculated as for AIMP3.</p

Potential binding regions mapped onto the crystal structure of AIMP3.

<p>Upper left: Interface I in yellow, including residues Arg⁵⁰, Thr⁶⁸, Lys⁷⁵, Ala⁹¹, Gln⁹⁴, Gln⁹⁵, Glu⁹⁸, and Asp¹¹⁹. Upper right: Segment 91–96 with significant decrease in D-uptake, overlapping with putative binding interface I. Lower left: Putative binding Interface II in green, including residues Glu¹²⁵, Val¹²⁸, Tyr¹²⁹, Tyr¹³³, Leu¹⁶², Arg¹⁶⁶, and Phe¹⁸⁶, does not exhibit any D-uptake change. Lower right: Novel binding site including segments N₁₃₄FTLAD₁₃₉, and L₁₄₃YYGLHRFIV₁₅₂. Color codes for the residues represent Average Relative D-uptake difference (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181869#pone.0181869.e001" target="_blank">Eq 1</a>).</p

Constructs for analyzing the AIMP3-LmnA interaction.

<p>A. Primary structure difference between mature LmnA and LmnC. B. Recombinant protein constructs for interaction analysis.</p