Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future. Molecular & Cellular

Abnormal expansions of an intronic hexanucleotide GGGGCC (G4C2) repeat of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies suggested that the C9orf72 hexanucleotide repeat expansion (HRE), either as DNA or the transcribed RNA, can fold into G-quadruplexes with distinct structures. These structural polymorphisms lead to abortive transcripts and contribute to the pathogenesis of ALS and FTD. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, we analyzed the structures of C9orf72 HRE DNA with various G4C2 repeats. They exhibited diverse G-quadruplex folds in potassium ions. Furthermore, we determined the topology of a G-quadruplex formed by d(G4C2)4. It favors a monomeric fold and forms a chair-type G-quadruplex with a four-layer antiparallel G-tetra core and three edgewise loops, which is distinct from known structures of chair-type G-quadruplexes. Our findings highlight the conformational heterogeneity of C9orf72 HRE DNA, and may lay the necessary structural basis for designing small molecules for the modulation of ALS/FTD pathogenesis.

The minichromosome maintenance (Mcm) 2-7 complex is the replicative helicase in eukaryotic species, and it plays essential roles in the initiation and elongation phases of DNA replication. During late M and early G 1 , the Mcm2-7 complex is loaded onto chromatin to form prereplicative complex in a Cdt1-dependent manner. However, the detailed molecular mechanism of this loading process is still elusive. In this study, we demonstrate that the previously uncharacterized C-terminal domain of human Mcm6 is the Cdt1 binding domain (CBD) and present its high resolution NMR structure. The structure of CBD exhibits a typical "winged helix" fold that is generally involved in protein-nucleic acid interaction. Nevertheless, the CBD failed to interact with DNA in our studies, indicating that it is specific for protein-protein interaction. The CBDCdt1 interaction involves the helix-turn-helix motif of CBD. The results reported here provide insight into the molecular mechanism of Mcm2-7 chromatin loading and prereplicative complex assembly.For the maintenance of genetic integrity, initiation of eukaryotic DNA replication is tightly controlled to ensure that DNA replication occurs exactly once in each cell cycle. Replication begins by the formation of pre-RCs 4 on replication origins during late M and G 1 phases (1, 2). For pre-RC assembly, the sixsubunit origin recognition complex first binds replication origin on newly synthesized chromatin. The origin recognition complex serves as an origin marker and recruits the initiation factors Noc3p, Cdc6, and Cdt1 to origins for the chromatin loading of the heterohexameric Mcm2-7 complex (3-5). Once the Mcm complex is loaded onto chromatin and pre-RC is formed, the cell is licensed for DNA replication, awaiting additional signals for the activation of the licensed origins (6). The Mcm2-7 complex was first identified as a set of genes required for minichromosome maintenance in budding yeast ( Cdt1 is a critical member of pre-RC, and its main function is to load Mcm2-7 helicase onto chromatin to license the DNA for replication in the subsequent S phase (16). Overexpression of Cdt1 alone in many types of mammalian cells is sufficient to induce rereplication (17-19). Previous studies have broadly defined three functional domains of Cdt1: a domain in the middle of the molecule containing the major Geminin interaction site; an N-terminal domain, which is required for ubiquitin-mediated proteolysis and contains a second interaction site for Geminin; and a C-terminal domain, which is required for association with Mcm proteins (12). Interactions between Cdt1 and individual members of the Mcm2-7 complex have been examined, and Cdt1 was found to interact with Mcm2 and Mcm6 (16,[21][22][23]. The existence of a stoichiometric complex between Cdt1 and Mcm2-7 was recently reported, which is consistent with earlier biochemical and genetic investigations (24). However, the detailed molecular mechanism underlying the chromatin loading of the Mcm2-7 complex through Cdt1 remains elusive.In this report, w...