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. After defining distinct stability properties of aspartate and glutamate ADP-ribosylation, we devise methods to install homogenous ADP-ribose chains at specific glutamate sites and assemble glutamate-modified peptides into full-length proteins. By implementing these technologies, we show that histone H2B E2 tri-ADP-ribosylation is able to stimulate the chromatin remodeler ALC1 with similar efficiency to histone serine ADP-ribosylation. Our work reveals fundamental principles of aspartate and glutamate ADP-ribosylation and enables new strategies to interrogate the biochemical consequences of this widespread protein modification.

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-ß structures. Mechanistic studies useful for the study of LCD self-association have evolved over the past decade in the form of simple assays of phase separation. Here, we have used such assays to demonstrate that the interactions responsible for LCD self-association can be dictated by labile protein structures poised close to equilibrium between the folded and unfolded states. Furthermore, missense mutations causing Charcot-Marie-Tooth disease, frontotemporal dementia, and Alzheimer's disease manifest their pathophysiology in vitro and in cultured cell systems by enhancing the stability of otherwise labile molecular structures formed upon LCD self-association.

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. We found that serine mono-ADP-ribosylation is sufficient to alleviate linker histone-dependent chromatin compaction and that this effect is amplified by ADP-ribose chain elongation. Our work will greatly expand the scope of ADP-ribose-modified proteins that can be constructed via semisynthesis, which is rapidly emerging as a robust approach to elucidate the direct effects that site-specific serine mono- and poly-ADP-ribosylation have on protein function.

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. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at histone H2B serine 6 or histone H3 serine 10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

IID572: A New Potentially Best-In-Class ß-Lactamase Inhibitor.

Resistance in Gram-negative bacteria to ß-lactam drugs is mediated primarily by the expression of ß-lactamases, and co-dosing of ß-lactams with a ß-lactamase inhibitor (BLI) is a clinically proven strategy to address resistance. New ß-lactamases that are not impacted by existing BLIs are spreading and creating the need for development of novel broader spectrum BLIs. IID572 is a novel broad spectrum BLI of the diazabicyclooctane (DBO) class that is able to restore the antibacterial activity of piperacillin against piperacillin/tazobactam-resistant clinical isolates. IID572 is differentiated from other DBOs by its broad inhibition of ß-lactamases and the lack of intrinsic antibacterial activity.

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae - Identification of LYS228.

Metallo-ß-lactamases (MBLs), such as New Delhi metallo-ß-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of ß-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine ß-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of ß-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).