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KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

Garg, Ankit; O'Rourke, Jason; Long, Chengzu; Doering, Jonathan; Ravenscroft, Gianina; Bezprozvannaya, Svetlana; Nelson, Benjamin R; Beetz, Nadine; Li, Lin; Chen, She; Laing, Nigel G; Grange, Robert W; Bassel-Duby, Rhonda; Olson, Eric N
Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40-/- mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients.
PMCID:4109545
PMID: 24960163
ISSN: 1558-8238
CID: 2271062

Partitioning of histone H3-H4 tetramers during DNA replication-dependent chromatin assembly

Xu, Mo; Long, Chengzu; Chen, Xiuzhen; Huang, Chang; Chen, She; Zhu, Bing
Semiconservative DNA replication ensures the faithful duplication of genetic information during cell divisions. However, how epigenetic information carried by histone modifications propagates through mitotic divisions remains elusive. To address this question, the DNA replication-dependent nucleosome partition pattern must be clarified. Here, we report significant amounts of H3.3-H4 tetramers split in vivo, whereas most H3.1-H4 tetramers remained intact. Inhibiting DNA replication-dependent deposition greatly reduced the level of splitting events, which suggests that (i) the replication-independent H3.3 deposition pathway proceeds largely by cooperatively incorporating two new H3.3-H4 dimers and (ii) the majority of splitting events occurred during replication-dependent deposition. Our results support the idea that "silent" histone modifications within large heterochromatic regions are maintained by copying modifications from neighboring preexisting histones without the need for H3-H4 splitting events.
PMID: 20360108
ISSN: 1095-9203
CID: 2271052

Two Prp19-like U-box proteins in the MOS4-associated complex play redundant roles in plant innate immunity

Monaghan, Jacqueline; Xu, Fang; Gao, Minghui; Zhao, Qingguo; Palma, Kristoffer; Long, Chengzu; Chen, She; Zhang, Yuelin; Li, Xin
Plant Resistance (R) proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC) along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein-mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity.
PMCID:2709443
PMID: 19629177
ISSN: 1553-7374
CID: 2271042

Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events

Tang, Chi-Tai; Li, Shaojie; Long, Chengzu; Cha, Joonseok; Huang, Guocun; Li, Lily; Chen, She; Liu, Yi
Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events.
PMCID:2705601
PMID: 19506251
ISSN: 1091-6490
CID: 2271032

Heterogeneous nuclear ribonucleoprotein L Is a subunit of human KMT3a/Set2 complex required for H3 Lys-36 trimethylation activity in vivo

Yuan, Wen; Xie, Jingwei; Long, Chengzu; Erdjument-Bromage, Hediye; Ding, Xiaojun; Zheng, Yong; Tempst, Paul; Chen, She; Zhu, Bing; Reinberg, Danny
The presence of histone H3 lysine 36 methylation (H3K36me) correlates with actively transcribed genes. In yeast, histone H3K36me mediated by KMT3 (also known as Set2) recruits a histone deacetylase complex, Rpd3s, to ensure the fidelity of transcription initiation. We report the purification of human KMT3a (also known as HYPB or hSet2) complex and the identification of a novel, higher eukaryotic specific subunit, heterogeneous nuclear ribonucleoprotein L (HnRNP-L). Interestingly, although KMT3a has intrinsic activity in vitro, HnRNP-L is essential in vivo. Moreover, KMT3a generates mono-, di-, and trimethylated products in vitro, but RNA interference against KMT3a or HnRNP-L down-regulates exclusively the H3K36me3 mark in vivo
PMCID:2708867
PMID: 19332550
ISSN: 0021-9258
CID: 121302

A bacterial type III effector family uses the papain-like hydrolytic activity to arrest the host cell cycle

Yao, Qing; Cui, Jixin; Zhu, Yongqun; Wang, Guolun; Hu, Liyan; Long, Chengzu; Cao, Ran; Liu, Xinqi; Huang, Niu; Chen, She; Liu, Liping; Shao, Feng
Pathogenic bacteria deliver effector proteins into host cells through the type III secretion apparatus to modulate the host function. We identify a family of proteins, homologous to the type III effector Cif from enteropathogenic Escherichia coli, in pathogens including Yersinia, Photorhabdus, and Burkholderia that contain functional type III secretion systems. Like Cif, this family of proteins is capable of arresting the host cell cycle at G(2)/M. Structure of one of the family members, Cif homolog in Burkholderia pseudomallei (CHBP), reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad despite the lack of primary sequence identity. For CHBP and Cif, only the putative catalytic Cys is susceptible to covalent modification by E-64, a specific inhibitor of papain-like cysteine proteases. Unlike papain-like enzymes where the S2 site is the major determinant of cleavage-site specificity, CHBP has a characteristic negatively charged pocket occupying surface areas corresponding to the S1/S1' site in papain-like proteases. The negative charge is provided by a conserved aspartate, and the pocket best fits an arginine, as revealed by molecular docking analysis. Mutation analysis establishes the essential role of the catalytic triad and the negatively charged pocket in inducing cell cycle arrest in host cells. Our results demonstrate that bacterial pathogens have evolved a unique papain-like hydrolytic activity to block the normal host cell cycle progression.
PMCID:2656146
PMID: 19225106
ISSN: 0027-8424
CID: 371452

Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide

Mi, Shijun; Cai, Tao; Hu, Yugang; Chen, Yemiao; Hodges, Emily; Ni, Fangrui; Wu, Liang; Li, Shan; Zhou, Huanyu; Long, Chengzu; Chen, She; Hannon, Gregory J; Qi, Yijun
Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.
PMCID:2981139
PMID: 18342361
ISSN: 1097-4172
CID: 2271022

Protein kinase A and casein kinases mediate sequential phosphorylation events in the circadian negative feedback loop

Huang, Guocun; Chen, She; Li, Shaojie; Cha, Joonseok; Long, Chengzu; Li, Lily; He, Qiyang; Liu, Yi
Regulation of circadian clock components by phosphorylation plays essential roles in clock functions and is conserved from fungi to mammals. In the Neurospora circadian negative feedback loop, FREQUENCY (FRQ) protein inhibits WHITE COLLAR (WC) complex activity by recruiting the casein kinases CKI and CKII to phosphorylate the WC proteins, resulting in the repression of frq transcription. On the other hand, CKI and CKII progressively phosphorylate FRQ to promote FRQ degradation, a process that is a major determinant of circadian period length. Here, by using whole-cell isotope labeling and quantitative mass spectrometry methods, we show that the WC-1 phosphorylation events critical for the negative feedback process occur sequentially-first by a priming kinase, then by the FRQ-recruited casein kinases. We further show that the cyclic AMP-dependent protein kinase A (PKA) is essential for clock function and inhibits WC activity by serving as a priming kinase for the casein kinases. In addition, PKA also regulates FRQ phosphorylation, but unlike CKI and CKII, PKA stabilizes FRQ, similar to the stabilization of human PERIOD2 (hPER2) due to the phosphorylation at the familial advanced sleep phase syndrome (FASPS) site. Thus, PKA is a key clock component that regulates several critical processes in the circadian negative feedback loop.
PMCID:2113029
PMID: 18079175
ISSN: 0890-9369
CID: 2271012

Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase

Zhu, Yongqun; Li, Hongtao; Long, Chengzu; Hu, Liyan; Xu, Hao; Liu, Liping; Chen, She; Wang, Da-Cheng; Shao, Feng
The OspF family of phosphothreonine lyase, including SpvC from Salmonella, irreversibly inactivates the dual-phosphorylated host MAPKs (pT-X-pY) through beta elimination. We determined crystal structures of SpvC and its complex with a phosphopeptide substrate. SpvC adopts a unique fold of alpha/beta type. The disordered N terminus harbors a canonical D motif for MAPK substrate docking. The enzyme-substrate complex structure indicates that recognition of the phosphotyrosine followed by insertion of the threonine phosphate into an arginine pocket places the phosphothreonine into the enzyme active site. This requires the conformational flexibility of pT-X-pY, which suggests that p38 (pT-G-pY) is likely the preferred physiological substrate. Structure-based biochemical and enzymatic analysis allows us to propose a general acid/base mechanism for beta elimination reaction catalyzed by the phosphothreonine lyase. The mechanism described here provides a structural understanding of MAPK inactivation by a family of pathogenic effectors conserved in plant and animal systems and may also open a new route for biological catalysis.
PMID: 18060821
ISSN: 1097-2765
CID: 371472

A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-induced immunity in plants

Zhang, Jie; Shao, Feng; Li, Yan; Cui, Haitao; Chen, Linjie; Li, Hongtao; Zou, Yan; Long, Chengzu; Lan, Lefu; Chai, Jijie; Chen, She; Tang, Xiaoyan; Zhou, Jian-Min
Pathogen-associated molecular patterns (PAMPs) elicit basal defense responses in plants, and, in turn, pathogens have evolved mechanisms to overcome these PAMP-induced defenses. To suppress immunity, the phytopathogenic bacterium Pseudomonas syringae secretes effector proteins, the biochemical function and virulence targets of which remain largely unknown. We show that HopAI1, an effector widely conserved in both plant and animal bacterial pathogens, inhibits the Arabidopsis mitogen-activated protein kinases (MAPKs) activated by exposure to PAMPs. HopAI1 inactivates MAPKs by removing the phosphate group from phosphothreonine through a unique phosphothreonine lyase activity, which is required for HopAI1 function. The inhibition of MAPKs by HopA1 suppresses two independent downstream events, namely the reinforcement of cell wall defense and transcriptional activation of PAMP response genes. The MAPKs MPK3 and MPK6 physically interact with HopAI1 indicating that they are direct targets of HopAI1. These findings uncover a mechanism by which Pseudomonas syringae overcomes host innate immunity to promote pathogenesis.
PMID: 18005697
ISSN: 1934-6069
CID: 2271002