Identification of potent and reversible piperidine carboxamides that are species-selective orally active proteasome inhibitors to treat malaria.

Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome β5 active-site (Pfβ5).

Enigmatic missense mutations can cause disease via creation of nuclear export signals.

Disease-causing missense mutations that occur within structurally and functionally unannotated protein regions can guide researchers to new mechanisms of protein regulation and dysfunction. Here, we report that the thrombocytopenia-, myelodysplastic syndromes-, and leukemia-associated P214L mutation in the transcriptional regulator ETV6 creates an XPO1-dependent nuclear export signal to cause protein mislocalization. Strategies to disrupt XPO1 activity fully restore ETV6 P214L protein nuclear localization and transcription regulation activity.

Asymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1.

The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break.

Chemoenzymatic and Synthetic Approaches To Investigate Aspartate- and Glutamate-ADP-Ribosylation.

We report here chemoenzymatic and fully synthetic methodologies to modify aspartate and glutamate side chains with ADP-ribose at specific sites on peptides. Structural analysis of aspartate and glutamate ADP-ribosylated peptides reveals near-quantitative migration of the side chain linkage from the anomeric carbon to the 2″- or 3″-ADP-ribose hydroxyl moieties. We find that this linkage migration pattern is unique to aspartate and glutamate ADP-ribosylation and propose that the observed isomer distribution profile is present in biochemical and cellular environments.

Mutations linked to neurological disease enhance self-association of low-complexity protein sequences.

Protein domains of low sequence complexity do not fold into stable, three-dimensional structures. Nevertheless, proteins with these sequences assist in many aspects of cell organization, including assembly of nuclear and cytoplasmic structures not surrounded by membranes. The dynamic nature of these cellular assemblies is caused by the ability of low-complexity domains (LCDs) to transiently self-associate through labile, cross-β structures.

A Protein Semisynthesis-Based Strategy to Investigate the Functional Impact of Linker Histone Serine ADP-Ribosylation.

Recently developed chemical and enzyme-based technologies to install serine ADP-ribosylation onto synthetic peptides have enabled new approaches to study poly(ADP-ribose) polymerase (PARP) biology. Here, we establish a generalizable strategy to prepare ADP-ribosylated peptides that are compatible with N-terminal, C-terminal, and sequential protein ligation reactions. Two unique protein-assembly routes are employed to generate full-length linker histone constructs that are homogeneously ADP-ribosylated at known DNA damage-dependent modification sites.

Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling.

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here, we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1.

Transiently structured head domains control intermediate filament assembly.

Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, β-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order.

A basic motif anchoring ISWI to nucleosome acidic patch regulates nucleosome spacing.

Recent studies have implicated the nucleosome acidic patch in the activity of ATP-dependent chromatin remodeling machines. We used a photocrosslinking-based nucleosome profiling technology (photoscanning) to identify a conserved basic motif within the catalytic subunit of ISWI remodelers, SNF2h, which engages this nucleosomal epitope. This region of SNF2h is essential for chromatin remodeling activity in a reconstituted biochemical system and in cells.

Nucleic Acid-Barcoding Technologies: Converting DNA Sequencing into a Broad-Spectrum Molecular Counter.

The emergence of high-throughput DNA sequencing technologies sparked a revolution in the field of genomics that has rippled into many branches of the life and physical sciences. The remarkable sensitivity, specificity, throughput, and multiplexing capacity that are inherent to parallel DNA sequencing have since motivated its use as a broad-spectrum molecular counter. A key aspect of extrapolating DNA sequencing to non-traditional applications is the need to append nucleic-acid barcodes to entities of interest.

Acetylation blocks DNA damage-induced chromatin ADP-ribosylation.

Recent studies report serine ADP-ribosylation on nucleosomes during the DNA damage response. We unveil histone H3 serine 10 as the primary acceptor residue for chromatin ADP-ribosylation and find that specific histone acetylation marks block this activity. Our results provide a molecular explanation for the well-documented phenomenon of rapid deacetylation at DNA damage sites and support the combinatorial application of PARP and HDAC inhibitors for the treatment of PARP-dependent cancers.

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system.

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells.

Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase ESCO1.

Protein acetylation is a prevalent posttranslational modification that is regulated by diverse acetyltransferase enzymes. Although histone acetyltransferases (HATs) have been well characterized both structurally and mechanistically, far less is known about non-histone acetyltransferase enzymes. The human ESCO1 and ESCO2 paralogs acetylate the cohesin complex subunit SMC3 to regulate the separation of sister chromatids during mitosis and meiosis.

A two-state activation mechanism controls the histone methyltransferase Suv39h1.

Specialized chromatin domains contribute to nuclear organization and regulation of gene expression. Gene-poor regions are di- and trimethylated at lysine 9 of histone H3 (H3K9me2 and H3K9me3) by the histone methyltransferase Suv39h1. This enzyme harnesses a positive feedback loop to spread H3K9me2 and H3K9me3 over extended heterochromatic regions.

The molecular basis for histone H4- and H2A-specific amino-terminal acetylation by NatD.

N-terminal acetylation is among the most common protein modifications in eukaryotes and is mediated by evolutionarily conserved N-terminal acetyltransferases (NATs). NatD is among the most selective NATs; its only known substrates are histones H4 and H2A, containing the N-terminal sequence SGRGK in humans. Here we characterize the molecular basis for substrate-specific acetylation by NatD by reporting its crystal structure bound to cognate substrates and performing related biochemical studies.

Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects.

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15.

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog.

Amino-terminal acetylation is a ubiquitous modification in eukaryotes that is involved in a growing number of biological processes. There are six known eukaryotic amino-terminal acetyltransferases (NATs), which are differentiated from one another on the basis of substrate specificity. To date, two eukaryotic NATs, NatA and NatE, have been structurally characterized, of which NatA will acetylate the α-amino group of a number of nonmethionine amino-terminal residue substrates such as serine; NatE requires a substrate amino-terminal methionine residue for activity.

Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.

N-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this cotranslational modification, the heterodimeric NatA complex has the most diversity for substrate selection and modifies the majority of all N-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100-kDa holo-NatA complex from Schizosaccharomyces pombe, in the absence and presence of a bisubstrate peptide-CoA-conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit.

Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs).

Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation.

The co-translational modification of N-terminal acetylation is ubiquitous among eukaryotes and has been reported to have a wide range of biological effects. The human N-terminal acetyltransferase (NAT) Naa50p (NAT5/SAN) acetylates the α-amino group of proteins containing an N-terminal methionine residue and is essential for proper sister chromatid cohesion and chromosome condensation. The elevated activity of NATs has also been correlated with cancer, making these enzymes attractive therapeutic targets.

Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor.

The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646.