Biology subjects

Dong, M.-Q.

Publications and source records attributed to Dong, M.-Q..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Somatic nuclear blebbing may indicate proliferating germ cells but does not indicate organismal aging in Caenorhabditis elegans

Abnormal nuclear morphology is suggested to be a hallmark of aging. One type of such abnormalities is nuclear blebbing, but little is known about whether and how nuclear blebbing participates in animal aging. What regulates nuclear blebbing is also unknown. In this study, we show that the frequency of nuclear blebbing in the hypodermis increases during aging in wild-type C. elegans. These nuclear blebs are enveloped by the nuclear lamina, the inner and the outer nuclear membrane, and 42% of them contain chromatin. Detachment of a bleb from the nucleus is rare but does happen, thereby generating cytoplasmic chromatin. Cytoplasmic chromatin-containing lysosomes juxtaposing the nucleus are detected in old worms. Therefore, nuclear blebbing contributes to the age-associated chromatin loss. However, the frequency of nuclear blebbing does not correlate with the rate of aging in C. elegans. Old age does not necessarily induce nuclear blebbing, neither does starvation, heat stress, or oxidative stress. Intriguingly, we find that proliferation of germ cells promotes nuclear blebbing. HighlightsO_LINuclear blebs accumulate in the hypodermis during C. elegans aging C_LIO_LINuclear blebbing contributes to chromatin loss C_LIO_LIThe frequency of nuclear blebbing does not correlate with the rate of aging C_LIO_LIProliferating germ cells promote nuclear blebbing in the hypodermis C_LI

cell biology↗

bioRxiv2022

PLK1 O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma

The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification, whose function and substrates have entranced biologists and chemists alike. O-GlcNAcylation occurs on Ser/Thr residues and takes part in a vast array of physiological processes. OGT is essential for dividing mammalian cells, and it underscores many human diseases. Yet many of its fundamental substrates in the cell division process remains to be unveiled. Here we focus on its effect on Polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation and mitotic exit. We found that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (MS) and mutagenesis studies, the critical O-GlcNAc site is located to be Thr291. Interestingly, T291N is a uterine carcinoma mutant in the TCGA database. Biochemical assays show that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we show that PLK1-T291A and -T291N mutants display chromosome segregation defects, and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and -T291N mutants would enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, and sceHCD MS might be a new method to reveal many more O-GlcNAcylation substrates.

cancer biology↗

bioRxiv2022

Old Age Drives Fusion and Expansion of Vitellogenin Vesicles in the Intestine of Caenorhabditis elegans

Some of the most conspicuous aging phenotypes of C. elegans are related to post-reproductive production of vitellogenins (Vtg), which form yolk protein (YP) complexes after processing and lipid loading. Vtg/YP levels show huge increases with age, and inhibition of this extends lifespan, but how subcellular and organism-wide distribution of these proteins changes with age has not been systematically explored. Here this has been done to understand how vitellogenesis promotes aging. The age-associated changes of intestinal vitellogenin vesicles (VVs), pseudocoelomic yolk patches (PYPs), and gonadal yolk organelles (YOs) have been characterized by immuno-electron microscopy. We find that from reproductive adult day 2 (AD 2) to post-reproductive AD 6 and AD 9, intestinal VVs expand from 0.2 m to 3-4 m in diameter or by > 3,000 times in volume, PYPs increase by > 3 times in YP concentration and volume, while YOs in oocytes shrink slightly from 0.5 m to 0.4 m in diameter or by 60% in volume. In AD 6 and AD 9 worms, mis-localized YOs found in the hypodermis, uterine cells, and the somatic gonadal sheath can reach a size of 10 m across in the former two tissues. This remarkable size increase of VVs and that of mis-localized YOs in post-reproductive worms are accompanied by extensive fusion between these Vtg/YP-containing vesicular structures in somatic cells. In contrast, no fusion is seen between YOs in oocytes. We propose that in addition to the continued production of Vtg, excessive fusion between VVs and mislocalized YOs in the soma worsen the aging pathologies seen in C. elegans. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/501051v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@cfda97org.highwire.dtl.DTLVardef@180369org.highwire.dtl.DTLVardef@87eedaorg.highwire.dtl.DTLVardef@525384_HPS_FORMAT_FIGEXP M_FIG Graphical Summary: Age-dependent changes of vitellogenins-/yolk proteins-containing subcellular structures in C. elegans On the left is a young wild-type C. elegans of adult day 1 or 2, and on the right is an older worm of adult day 6 or 9. The orange-colored structures are: in the intestine, vitellogenin vesicles (VVs), of a median diameter of 200 nm on the left and 3-4 micrometers on the right, with a frequent fusion between VVs in the older worm; in the pseudocoelom, pseudocoelomic yolk patches, greatly expanded in the older worm; in oocytes and eggs, yolk organelles (YOs), of a median diameter of 500 nm on the left and 400 nm on the right; in the hypodermis, sheath or uterine cells of the older worm, mislocalized YOs. C_FIG

cell biology↗

bioRxiv2022

Collisional dissociation of 115 deprotonated peptides of 5-28 amino acids--an updated report on fragment ion types, neutral loss species, and the effect of acidic residues

Highly acidic, D/E-rich peptides or proteins are difficult to identify by positive-ion-mode mass spectrometry (MS), and negative-ion-mode MS is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant backbone or side-chain neutral losses (NL), and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y- > x-, z- > c- if the NL ions are included, or c- > y- > z- > b- if not. The most frequent side-chain NLs involve amino acids C, S, T, D, E, N, Q, and R. Although NL of CO2 is common for all peptides, it is markedly enhanced in D/E-containing peptides. Long peptides and D/E-rich peptides are prone to carrying multiple negative charges. HCD spectra produced from multiply deprotonated peptides have a lower fraction of sequencing ions (i.e., a-, b-, c-, x-, y-, z- ions) than those produced from 1- ([M-H]-) and 2- ([M-2H]2-) precursors. Based on the above findings, we predict that under negative-ion HCD, D/E-rich peptides should be difficult to identify and that choosing a protease to generate peptides containing fewer D/E residues will improve identification of highly acidic proteins.

biochemistry↗

bioRxiv2022

Immuno-electron microscopy localizes Caenorhabditis elegans vitellogenins along the classic exocytosis route

Vitellogenins (VITs) are the most abundant proteins in adult hermaphrodite C. elegans. VITs are synthesized in the intestine, secreted to the pseudocoelom, matured into yolk proteins (YPs), and finally deposited in oocytes to support embryonic and larval development. How VITs are secreted out of the intestine remains unclear. In this study, we use immuno-electron microscopy (immuno-EM) to characterize the wild-type subcellular structures containing VITs or YPs. In the intestinal cells of young adult worms, we identify VITs along an exocytic pathway consisting of the rough ER, the Golgi, and the lipid bilayer bounded vesicles, which we call intestinal vitellogenin vesicles (VVs). This suggests that the classic exocytotic pathway mediates secretion of VITs from the intestine to the pseudocoelom. We also show that pseudocoelomic yolk patches (PYPs) are membrane-less and amorphous. The different VITs/YPs are packed as a mixture into the above structures. The size of VVs can vary with the VIT levels and the age of the worm. On adult day 2 (AD 2), intestinal VVs ([~]200 nm in diameter) are smaller than gonadal yolk organelles (YOs, [~]500 nm in diameter). VVs, PYPs, and YOs share a uniform, medium electron density by conventional EM. The morphological profiles documented in this study can serve as a reference for future studies of VITs/YPs. Surveying the findings from this study and elsewhere, we review in the discussion the post-translational modifications and protein-protein interactions of C. elegans VITs/YPs.

cell biology↗

bioRxiv2022

Preferential cross-linking of the stereospecific complex over the encounter complexes by DOPA2, a faster cross-linker than DSS

Transient protein-protein interactions are fundamental aspects of many biochemical reactions, but they are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) analysis is a powerful tool to facilitate the analysis of transient interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes--EIN/HPr with a KD of 7 M and EIIAGlc/EIIBGlc with a KD of 25 M--as model systems, we compared the effects of two amine-specific homo-bifunctional cross-linkers of different cross-linking speeds. Protein cross-linking by DOPA2, a di-ortho-phthalaldehyde cross-linker, is 60-120 times faster than that by DSS, an N-hydroxysuccinimide ester cross-linker. We analyzed the differences in the number of cross-links identified that reflected the stereospecific complex (SC), the final lowest-energy conformational state, and that of cross-links that reflected the encounter complexes (ECs), an ensemble of short-lived intermediate conformations mediated by nonspecific electrostatic interactions. We found that the faster DOPA2 cross-linking favored the SC whereas the slower DSS cross-linking favored the ECs. We propose a mechanistic model for this intriguing observation. This study suggests that it is feasible to probe the dynamics of protein-protein interaction using cross-linkers of different cross-linking speeds.

biochemistry↗

bioRxiv2022

The endoplasmic reticulum membrane protein complex (EMC) negatively regulates intestinal homeostasis through the Hippo signaling pathway

Disruption of tissue homeostasis often results in many diseases. Balanced control of stem cell proliferation and differentiation underlines tissue homeostasis. However, how endogenous factors influence the proliferation and differentiation of intestinal stem cells (ISCs) under physiological conditions remains not fully understood. Here, we find that the endoplasmic reticulum membrane protein complex (EMC) negatively regulates ISC proliferation in adult Drosophila midgut. Compromising EMC function in progenitors leads to excessive ISC proliferation and intestinal homeostasis disruption. Mechanistically, the EMC complex associates with and stabilizes Hippo (Hpo), the key component of the Hpo signaling pathway. In the absence of the EMC complex, Yki (Yorkie) is activated to promote ISC proliferation. Furthermore, the role of the EMC complex in stem cell proliferation control is evolutionarily conserved. Thus, our study uncovers the molecular mechanism of the EMC complex in controlling stem cell proliferation. Our results provide new insight into the underlying mechanisms of how stem cell proliferation is properly controlled under physiological conditions.

cell biology↗

bioRxiv2022

A family of C. elegans VASA homologs control Argonaute pathway specificity and promote transgenerational silencing

Germline Argonautes direct transcriptome surveillance within peri-nuclear membraneless organelles called nuage. In C. elegans, a family of Vasa-related Germ Line Helicase (GLH) proteins localize in, and promote the formation of nuage called P granules. Previous studies have implicated GLH proteins in inherited silencing but direct roles in amplification of small RNAs, or in target mRNA or Argonatue binding have not been identified. Here we show that GLH proteins compete with each other to control Argonaute pathway specificity, bind directly to Argonaute-target mRNAs and act to promote the amplification of small RNAs required for transgenerational inheritance. We show that the ATPase cycle of GLH-1 regulates its direct binding to the Argonaute WAGO-1 which engages amplified small RNAs. Our findings support a dynamic and direct role for GLH proteins in inherited silencing beyond their role as structural components of nuage.

molecular biology↗

bioRxiv2021

Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32

Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and have three forms- TRAPPI, TRAPPII and TRAPPIII, which share a core of six TRAPPI proteins. TRAPPII facilitates intra-Golgi and endosome-to-Golgi transports by activating GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor (GEF) in yeast. Here we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle shaped monomers, while the monomer in the apo structure exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation. Located in the interior of the monomer, Ypt32 binds with both TRAPPI and Trs120 via its nucleotide binding domain and binds with Trs31 of TRAPPI via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII. One Sentence SummaryStructures of TRAPPII in different states reveal the mechanism of the specific activation of Ypt32 by TRAPPII.

biophysics↗

bioRxiv2021

Degrading intestinal DAF-2 nearly doubles Caenorhabditis elegans lifespan without affecting development or reproduction

Twenty-eight years following the breakthrough discovery that a single-gene mutation of daf-2 can double the lifespan of Caenorhabditis elegans, it remains unclear where this gene, which encodes an insulin/IGF-1 receptor, is expressed and where it acts to regulate aging. Here, by inserting DNA sequences of fluorescent tags into the genomic locus of daf-2 and that of its downstream transcription factor daf-16, we determined that both genes are expressed in most or all tissues from embryos through adulthood, in line with their diverse functions. Using tissue-specific auxin-induced protein degradation, we determined that both DAF-2 and DAF-16 act in the intestine to regulate organismal aging. Strikingly, loss of DAF-2 in the intestine nearly doubled C. elegans lifespan but did not produce the adverse developmental or reproductive phenotypes associated with genetic daf-2 mutants. These findings unify the mechanism of lifespan regulation by genes and that by dietary restriction, and begin to focus anti-aging research on nutrient supply. HighlightsO_LIdaf-2 and daf-16 are expressed in most or all cells of C. elegans using genome editing. C_LIO_LIDAF-2 and DAF-16 both regulate lifespan from the intestine as determined using auxin-induced protein degradation. C_LIO_LIReduced insulin signaling in the intestine nearly doubles C. elegans lifespan without adverse effects on development or reproduction. C_LIO_LILifespan regulation by genes and dietary restriction are unified by intestinal supply of nutrients and metabolism. C_LI

cell biology↗

bioRxiv2021

Molecular and structural mechanisms of ZZ domain-mediated cargo recognition by autophagy receptor Nbr1

In selective autophagy, cargo selectivity is determined by autophagy receptors. However, it remains scarcely understood how autophagy receptors recognize specific protein cargos. In the fission yeast Schizosaccharomyces pombe, a selective autophagy pathway termed Nbr1-mediated vacuolar targeting (NVT) employs Nbr1, an autophagy receptor conserved across eukaryotes including humans, to target cytosolic hydrolases into the vacuole. Here, we identify two new NVT cargos, the mannosidase Ams1 and the aminopeptidase Ape4, that bind competitively to the first ZZ domain of Nbr1 (Nbr1-ZZ1). High-resolution cryo-EM analyses reveal how a single ZZ domain recognizes two distinct protein cargos. Nbr1-ZZ1 not only recognizes the N-termini of cargos via a conserved acidic pocket, similar to other characterized ZZ domains, but also engages additional parts of cargos in a cargo-specific manner. Our findings unveil a single-domain bispecific mechanism of autophagy cargo recognition, elucidate its underlying structural basis, and expand the understanding of ZZ domain-mediated protein-protein interactions.

cell biology↗

bioRxiv2020

A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.

biochemistry↗

bioRxiv2020

PIE-1 promotes SUMOylation and activation of HDAC1 during the C. elegans oogenesis

Germlines shape and balance heredity, integrating and regulating information from both parental and foreign sources. Insights into how the germline handles information have come from the identification of factors that specify or maintain the germline fate. In early C. elegans embryos, the CCCH zinc-finger protein PIE-1 localizes to the germline where it prevents somatic differentiation programs. Here we show that PIE-1 also functions in the meiotic ovary where it becomes SUMOylated and engages the SUMO-conjugating machinery. Using whole-proteome mass spectrometry to detect SUMO-conjugated proteins, we identify HDAC SUMOylation as a target of PIE-1. Our findings suggest that SUMOylation activates HDAC, lowering histone acetylation and enhancing Argonaute-mediated surveillance in the germline.

molecular biology↗

bioRxiv2020

HDAC1 SUMOylation promotes Argonaute directed transcriptional silencing in C. elegans

Eukaryotic cells use guided search to coordinately control dispersed genetic elements. The transitive effectors of these mechanisms, Argonaute proteins and their small-RNA co-factors, engage nascent RNAs and chromatin-associated proteins to direct transcriptional silencing. The small ubiquitin-like modifier (SUMO) has been shown to promote the induction and maintenance of silent chromatin (called heterochromatin) in yeast, plants, and animals. Here we show that Argonaute-directed transcriptional silencing in C. elegans requires SUMOylation of the type 1 histone deacetylase HDA-1. SUMOylation of HDA-1 promotes interactions with components of the nucleosome remodeling and deacetylase (NuRD) complex and with the nuclear Argonaute HRDE-1/WAGO-9. Our findings suggest how HDAC1 SUMOylation promotes the association of HDAC and other chromatin remodeling factors with a nuclear Argonaute in order to initiate de novo heterochromatin silencing.

molecular biology↗

bioRxiv2020

Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation

Molecular glues are small molecules that exert their biologic or therapeutic activities by inducing gain-of-function interactions between pairs of proteins. In particular, molecular-glue degraders, which mediate interactions between target proteins and components of the ubiquitin proteasome system to cause targeted protein degradation, hold great promise as a unique modality for therapeutic targeting of proteins that are currently intractable. Here, we report a new molecular glue HQ461 discovered by high-throughput screening of small molecules that inhibited NRF2 activity. Using unbiased loss-of-function and gain-of-function genetic screening followed by biochemical reconstitution, we show that HQ461 acts by promoting interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of CDK12s interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that engages its target protein directly with DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.

biochemistry↗

bioRxiv2018

Differential phase partition of a PICS complex is required for piRNA processing and chromosome segregation in C. elegans

Piwi-interacting RNAs (piRNAs) play significant roles in suppressing transposons and non-self nucleic acids, maintaining genome integrity, defending against viral infections, and are essential for fertility in a variety of organisms. In C. elegans, most piRNA precursors are transcribed by RNA polymerase II in the nucleus and are subjected to a number of processing and maturation steps. However, the biogenesis of piRNAs are still not fully understood. We used functional proteomics to study piRNA biogenesis in C. elegans and identified a PICS complex that is required for piRNA processing and chromosome segregation. The PICS complex contains two known piRNA biogenesis factors TOFU-6 and PID-1, and three new proteins PICS-1, TOST-1, and ERH-2, which exhibit dynamic localization among different subcellular compartments. In the germline of gravid animals, the PICS complex contains TOFU-6/PICS-1/ERH-2/PID-1 and largely concentrate at the perinuclear granule zone and engages in piRNA processing. During early embryogenesis, the TOFU-6/PICS-1/ERH-2/TOST-1 complex accumulates in the nucleus and play essential roles in chromosome segregation and cell division. Interestingly, the function of these factors in mediating chromosome segregation is independent of piRNA production. Therefore, we speculate that a differential phase partition of PICS factors may help cells to coordinate distinct cellular processes.

genetics↗

bioRxiv2018

The USTC complex co-opts an ancient machinery to drive piRNA transcription in C. elegans

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and non-self nucleic acids, maintain genome integrity, and are essential for fertility in a variety of organisms. In C. elegans most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis the mechanism of piRNA transcription remains elusive. Here we used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis. The USTC complex contains PRDE-1, TOFU-4, TOFU-5 and SNPC-4. The USTC complex form a unique piRNA foci in germline nuclei and coat the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. USTC factors are also mutually dependent for binding to the piRNA clusters and to form the piRNA foci. Interestingly, USTC components bind differentially to piRNAs in the clusters and other non-coding RNA genes. These results reveal USTC as a striking example of the repurposing of a general transcription factor complex to aid in genome defence against transposons.

developmental biology↗

bioRxiv2018

Insulin Signaling-independent Activation of DAF-16 Shapes the Transcriptome during Normal Aging

The roles and regulatory mechanisms of transriptome changes during aging are unclear. It has been proposed that the transcriptome suffers decay during aging owing to age-associated down-regulation of transcription factors. In this study, we characterized the role of a transcription factor DAF-16, which is a highly conserved lifespan regulator, in the normal aging process of Caenorhabditis elegans. We found that DAF-16 translocates into the nucleus in aged wild-type worms and activates the expression of hundreds of genes in response to age-associated cellular stress. Most of the age-dependent DAF-16 targets are different from the canonical DAF-16 targets downstream of insulin signaling, indicating that activation of DAF-16 during aging is not due to reduced insulin signaling from DAF-2. Further analysis showed that it is due to the loss of proteostasis during aging, at least in part. We also found that without daf-16, dramatic gene expression changes occur as early as on adult day 2, indicating that DAF-16 acts to stabilize the transcriptome during normal aging. Our results thus reveal that normal aging is not simply a process in which the gene expression program descends into chaos due to loss of regulatory activities; rather, there is active transcriptional regulation that fights aging.

systems biology↗