Kevin E.W. Namitz's research while affiliated with Pennsylvania State University and other places

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

We performed a thorough analysis and characterization of multiple batches of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed at an industrial scale in a yeast system. Bottom-up LC-MS/MS-based proteomics analysis detected the full sequence of Helaina rhLF protein and confirmed that its amino acid sequence is identical to that of native human LF (Uniprot i.d. P02788). Helaina rhLF had a protein purity of 98% or higher as determined by three orthogonal methods; reversed-phase HPLC, SDS-PAGE, and LC-MS proteomics analysis. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642. The identified N-glycans of Helaina rhLF were predominantly oligomannose structures with five to nine mannoses (M5-M9), which we also report to be present in both the native human and bovine LF. human milk LF (hmLF) possessed lower levels of oligomannose structures and were mainly M5 and M6. Helaina rhLF protein secondary structure was nearly identical to that of hmLF, as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7+/-0.9 °C and 91.9+/-0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, the characterization analysis results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

Enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation are crucial for proper cellular differentiation and are frequently mutated in cancer. The Mixed Lineage leukemia (MLL) family of enzymes deposit H3K4 mono- di- or trimethylation at distinct genomic locations, requiring precise spatial and temporal control. Despite evidence that the degree of H3K4 methylation is controlled in part by a hierarchical assembly pathway with key subcomplex components, we previously found that the assembled state of the MLL1 core complex is not favored at physiological temperature. To better understand this paradox, we tested the hypothesis that increasing the concentration of subunits in a biomolecular condensate overcomes this thermodynamic barrier via mass action. Here we demonstrate that MLL1 core complex phase separation stimulates enzymatic activity up to 60-fold, but not primarily by concentrating subunits into droplets. Instead, we found that stimulated activity is largely due to formation of an altered oligomeric scaffold that greatly reduces substrate Km. We posit that phase separation induced scaffolding of the MLL1 core complex is a potential "switch-like" mechanism for spatiotemporal control of H3K4 methylation through the rapid formation or dissolution of biomolecular condensates within RNA Pol II transcription factories.

Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [1H, 13C]-HSQC and 13C direct-detect NMR measurements. Finally, we present 13C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes.

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g., K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolve the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

Enzymes of the Mixed Lineage Leukemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases are critical for cellular differentiation and development and are regulated by interaction with a conserved subcomplex consisting of WDR5, RbBP5, Ash2L, and DPY30 (WRAD2). While pairwise interactions between complex subunits have been determined, the mechanisms regulating holo-complex assembly are unknown. In this investigation, we systematically characterized the biophysical properties of a reconstituted human MLL1 core complex and found that the MLL1-WDR5 heterodimer interacts with the RbBP5-Ash2L-DPY30 subcomplex in a hierarchical assembly pathway that is highly dependent on concentration and temperature. Surprisingly, we found that the disassembled state is favored at physiological temperature, where the enzyme rapidly becomes irreversibly inactivated, likely due to complex components becoming trapped in non-productive conformations. Increased protein concentration partially overcomes this thermodynamic barrier for complex assembly, suggesting a potential regulatory mechanism for spatiotemporal control of H3K4 methylation. Together, these results are consistent with the hypothesis that regulated assembly of the MLL1 core complex underlies an important mechanism for establishing different H3K4 methylation states in mammalian genomes.