Adam G. Schwaid

Peptidomic discovery of short open reading frame–encoded peptides in human cells

The amount of the transcriptome that is translated into polypeptides is of fundamental importance. We developed a peptidomic strategy to detect short ORF (sORF)-encoded polypeptides (SEPs) in human cells. We identified 90 SEPs, 86 of which are novel, the largest number of human SEPs ever reported. SEP abundances range from 10-1000 molecules per cell, identical to known proteins. SEPs arise from sORFs in non-coding RNAs as well as multi-cistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that non-canonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8/1866) of long intergenic non-coding RNAs (lincRNAs). Together, these results provide the strongest evidence to date that the human proteome is more complex than previously appreciated.

Methods for the Elucidation of Protein-Small Molecule Interactions

Understanding the interactions between small molecules and proteins can be approached from different perspectives and is important for the advancement of basic science and drug development. Chemists often use bioactive small molecules, such as natural products or synthetic compounds, as probes to identify therapeutically relevant protein targets. Biochemists and biologists often begin with a specific protein and seek to identify the endogenous metabolites that bind to it. These interests have led to the development of methodology that relies heavily on synthetic and analytical chemistry to identify protein-small molecule and protein-metabolite interactions. Here, we survey these strategies, highlighting key findings, to demonstrate the value of these approaches in answering important chemical and biological questions.

Ligand activation of ERRα by cholesterol mediates statin and bisphosphonate effects

Nuclear receptors (NRs) are key regulators of gene expression and physiology. Nearly half of all human NRs lack endogenous ligands including estrogen-related receptor α (ERRα). ERRα has important roles in cancer, metabolism, and skeletal homeostasis. Affinity chromatography of tissue lipidomes with the ERRα ligand-binding domain (LBD) and subsequent transcriptional assays identified cholesterol as an endogenous ERRα agonist. Perturbation of cholesterol biosynthesis or inhibition of ERRα revealed the interdependence of cholesterol and ERRα. In bone, the effects of cholesterol, statin, and bisphosphonate on osteoclastogenesis require ERRα; and consequently, cholesterol-induced bone loss or bisphosphonate osteoprotection is lost in ERRα knockout mice. Furthermore, statin induction of muscle toxicity and cholesterol suppression of macrophage cytokine secretion are impaired by loss or inhibition of ERRα. These findings reveal a key step in ERRα regulation and explain the actions of two highly prescribed drugs, statins and bisphosphonates.

Adenine-DNA adducts derived from the highly tumorigenic Dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not.

The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N(2)-dG) and adenine (N(6)-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N(2)-dG linkage site is ∼35 times more susceptible to NER dual incisions than the stereochemically identical N(6)-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar but somewhat smaller effect (factor of ∼15) is observed. The striking resistance of the bulky N(6)-dA in contrast to the modest to good susceptibilities of the N(2)-dG adducts to NER is interpreted in terms of the balance between lesion-induced DNA distorting and DNA stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA.

Chemoproteomic Discovery of Cysteine-Containing Human Short Open Reading Frames

The application of ribosome profiling and mass spectrometry technologies has recently revealed that the human proteome is larger than previously appreciated. Short open reading frames (sORFs), which are difficult to identify using traditional gene-finding algorithms, constitute a significant fraction of unknown protein-coding genes. Thus, experimental approaches to identify sORFs provide invaluable insight into the protein-coding potential of genomes. Here, we report an affinity-based approach to enrich and identify cysteine-containing human sORF-encoded polypeptides (ccSEPs) from cells. This approach revealed 16 novel ccSEPs, each derived from an uncharacterized sORF, demonstrating its potential for discovering new genes. We validated expression of a SEP from its endogenous RNA, and demonstrated the specificity of our labeling approach using synthetic SEP. The discovery of additional human SEPs and their conservation indicate the potential importance of these molecules in biology.

A bulky DNA lesion derived from a highly potent polycyclic aromatic tumorigen stabilizes nucleosome core particle structure

The impact of a bulky DNA lesion on the structure and dynamics of a nucleosome core particle (NCP) containing a lesion derived from the unusually potent tumorigen dibenzo[a,l]pyrene that resists nucleotide excision repair (NER) in free DNA was investigated using 65 ns molecular dynamics simulations. Our results reveal that, relative to unmodified NCP, the lesion stabilizes the nucleosome via stacking interactions, improved Watson-Crick base pairing, hydrogen bonding between DNA and histones, and damped dynamics. These findings suggest that such lesions should be as resistant to NER in the nucleosome environment as they are in free DNA.

Free energy profiles of base flipping in intercalative polycyclic aromatic hydrocarbon-damaged DNA duplexes: energetic and structural relationships to nucleotide excision repair susceptibility.

The crystal structure of Rad4/Rad23, the yeast homolog of the human nucleotide excision repair (NER) lesion recognition factor XPC-RAD23B ( Min , J. H. and Pavletich , N. P. ( 2007 ) Nature 449 , 570 - 575 ) reveals that the lesion-partner base is flipped out of the helix and binds to amino acids of the protein. This suggests the hypothesis that the flipping of this partner base must overcome a free energy barrier, which constitutes one element contributing to changes in the thermodynamic properties induced by the DNA damage and sensed by the recognition protein. We explored this hypothesis by computing complete flipping free energy profiles for two lesions derived from the procarcinogenic polycyclic aromatic hydrocarbons (PAHs), dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P), R-trans-anti-DB[a,l]P-N(6)-dA (R-DB[a,l]P-dA) and R-trans-anti-B[a]P-N(6)-dA (R-B[a]P-dA), and the corresponding unmodified duplex. The DB[a,l]P and B[a]P adducts differ in number and organization of their aromatic rings. We integrate these results with prior profiles for the R-trans-anti-DB[a,l]P-dG adduct ( Zheng , H. et al. ( 2010 ) Chem. Res. Toxicol. 23 , 1868 - 1870 ). All adopt conformational themes involving intercalation of the PAH aromatic ring system into the DNA duplex; however, R-DB[a,l]P-dA and R-B[a]P-dA intercalate from the major groove, while R-DB[a,l]P-dG intercalates from the minor groove. These structural differences produce different computed van der Waals stacking interaction energies between the flipping partner base with the lesion aromatic ring system and adjacent bases; we find that the better the stacking, the higher the relative flipping free energy barrier and hence lower flipping probability. The better relative NER susceptibilities correlate with greater ease of flipping in these three differently intercalated lesions. In addition to partner base flipping, the Rad4/Rad23 crystal structure shows that a protein-β-hairpin, BHD3, intrudes from the major groove side between the DNA strands at the lesion site. We present a molecular modeling study for the R-DB[a,l]P-dG lesion in Rad4/Rad23 showing BHD3 β-hairpin intrusion with lesion eviction, and we hypothesize that lesion steric effects play a role in the recognition of intercalated adducts.

Maternal Adiponectin Controls Milk Composition to Prevent Neonatal Inflammation

Adiponectin is an important adipokine. Increasing evidence suggests that altered adiponectin levels are linked with metabolic and inflammatory disorders. Here we report an important yet previously unrecognized function of adiponectin in lactation by which maternal adiponectin determines the inflammatory status in the nursing neonates. Surprisingly, both maternal adiponectin overexpression in the transgenic mice and maternal adiponectin deletion in the knockout mice lead to systemic inflammation in the pups, manifested as transient hair loss. However, distinct mechanisms are involved. Adiponectin deficiency triggers leukocyte infiltration and production of inflammatory cytokines in the lactating mammary gland. In contrast, adiponectin overabundance increases lipid accumulation in the lactating mammary gland, resulting in excessive long-chain saturated fatty acids in milk. Interestingly, in both cases, the inflammation and alopecia in the pups can be rescued by Toll-like receptor (TLR)-2/4 deletion because TLR2/4 double-knockout pups are resistant. Mechanistically, long-chain saturated fatty acid activation of inflammatory genes is TLR2/4 dependent and can be potentiated by proinflammatory cytokines, indicating that the inflammatory stimuli in both scenarios functionally converge by activating the TLR2/4 signaling. Therefore, our findings reveal adiponectin as a dosage-dependent regulator of lactation homeostasis and milk quality that critically controls inflammation in the nursing neonates. Furthermore, these results suggest that inflammatory infantile disorders may result from maternal adiponectin dysregulation that can be treated by TLR2/4 inhibition.