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Biomedical subjects

Zhou Songyang

Publications and source records attributed to Zhou Songyang.

17 recordsLinked to original sources
PubMed2026

MYDGF as a telomerase activator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) involves progressive cartilage breakdown. This is driven by impaired chondrocyte function and an imbalance in the extracellular matrix homeostasis. However, the key upstream regulators that maintain healthy chondrocytes are still not fully known. Here, we report that myeloid-derived growth factor (MYDGF) is a novel regulator of telomerase activity and is critical for cartilage health. Using a genome-wide CRISPR-Cas9 screen with a TERT reporter system, MYDGF was identified as a strong positive regulator of telomerase. Functional studies confirmed that MYDGF positively regulates TERT expression and telomerase activity in both HeLa and ATDC5 cells. In a mouse surgical destabilization of the medial meniscus (DMM) OA model, MYDGF knockout (KO) mice exhibited more severe cartilage damage. Conversely, delivering MYDGF into the knee joint using AAV virus partially alleviated cartilage injury. Transcriptome profiling revealed that MYDGF-deficient chondrocytes exhibit downregulated key pathways involved in matrix building and upregulated inflammatory signals, indicating a shift in cell state. In summary, our work establishes MYDGF as a key upstream factor for both telomerase and cartilage matrix homeostasis remodeling. It connects telomerase function to chondrocyte health. These findings also highlight MYDGF as a promising new target for treating osteoarthritis.

Cartilage degeneration↗
PubMed2025

RNA-DNA hybrid binding domain broadens the editing window of base editors.

Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.

Gene Editing↗
PubMed2006

A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners.

We developed a retrovirus-based protein-fragment complementation assay (RePCA) screen to identify protein-protein interactions in mammalian cells. In RePCA, bait protein is fused to one fragment of a rationally dissected fluorescent protein, such as GFP, intensely fluorescent protein, or red fluorescent protein. The second, complementary fragment of the fluorescent protein is fused to an endogenous protein by in-frame exon traps in the enhanced retroviral mutagen vector. An interaction between bait and host protein (prey) places the two parts of the fluorescent molecule in proximity, resulting in reconstitution of fluorescence. By using RePCA, we identified a series of 24 potential interaction partners or substrates of the serine/threonine protein kinase AKT1. We confirm that alpha-actinin 4 (ACTN4) interacts physically and functionally with AKT1. siRNA-mediated ACTN4 silencing down-regulates AKT phosphorylation, blocks AKT translocation to the membrane, increases p27(Kip1) levels, and inhibits cell proliferation. Thus, ACTN4 is a critical regulator of AKT1 localization and function.

Actinin↗
PubMed2006

A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly.

Mammalian telomeric proteins function through dynamic interactions with each other and telomere DNA. We previously reported the formation of a high-molecular-mass telomeric complex (the mammalian telosome) that contains the six core proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 (formerly named PTOP/PIP1/TINT1) and mediates telomere end-capping and length control. In this report, we sought to elucidate the mechanism of six-protein complex (or shelterin) formation and the function of this complex. Through reconstitution experiments, we demonstrate here that TIN2 and TPP1 are key components in mediating the six-protein complex assembly. We demonstrate that not only TIN2 but also TPP1 are required to bridge the TRF1 and TRF2 subcomplexes. Specifically, TPP1 helps to stabilize the TRF1-TIN2-TRF2 interaction and promote six-protein complex formation. Consistent with this model, overexpression of TPP1 enhanced TIN2-TRF2 association. Conversely, knocking down TPP1 reduced the ability of endogenous TRF1 to associate with the TRF2 complex. Our results suggest that coordinated interactions among TPP1, TIN2, TRF1, and TRF2 may ensure robust assembly of the telosome, telomere targeting of its subunits, and, ultimately, regulated telomere maintenance.

Animals↗
PubMed2006

Inside the mammalian telomere interactome: regulation and regulatory activities of telomeres.

Work in model organisms, such as mouse, yeast, Tetrahymena, ciliates, and plants, has led to a deeper understanding of telomere biology. Telomeres together with telomere-binding proteins have evolved to protect chromosomal ends and maintain chromosomal length and integrity. Over the last two decades, biochemical, molecular, cellular, and genetic studies have greatly enhanced our knowledge of the unique function and structure of telomeres and telomere-associated factors. In this review, we focus on the important advances, in terms of our knowledge and the methods used, in understanding mammalian telomere regulation by telomeric proteins. Recently, the 6 telomeric proteins (TRF1, TRF2, POT1, TIN2, RAP1, and TPP1) were found to form a high-order complex. This complex and its associated partners provide the basis for constructing an interaction map of telomere regulators in mammalian cells, which we named the Telomere Interactome. The Telomere Interactome incorporates the various telomere signaling pathways and represents the molecular machinery that regulates mammalian telomeres. The establishment of the Telomere Interactome will also enable the integration of the intricate circuitries that regulate telomeres with other cellular interactomes in vertebrates.

Animals↗
PubMed2004

Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins.

In mammalian cells, telomere-binding proteins TRF1 and TRF2 play crucial roles in telomere biology. They interact with several other telomere regulators including TIN2, PTOP, POT1, and RAP1 to ensure proper maintenance of telomeres. TRF1 and TRF2 are believed to exert distinct functions. TRF1 forms a complex with TIN2, PTOP, and POT1 and regulates telomere length, whereas TRF2 mediates t-loop formation and end protection. However, whether cross-talk occurs between the TRF1 and TRF2 complexes and how the signals from these complexes are integrated for telomere maintenance remain to be elucidated. Through gel filtration and co-immunoprecipitation experiments, we found that TRF1 and TRF2 are in fact subunits of a telomere-associated high molecular weight complex (telosome) that also contains POT1, PTOP, RAP1, and TIN2. We demonstrated that the TRF1-interacting protein TIN2 binds TRF2 directly and in vivo, thereby bridging TRF2 to TRF1. Consistent with this multi-protein telosome model, stripping TRF1 off the telomeres by expressing tankyrase reduced telomere recruitment of not only TIN2 but also TRF2. These results help to unify previous observations and suggest that telomere maintenance depends on the multi-subunit telosome.

Blotting, Western↗
PubMed2004

PTOP interacts with POT1 and regulates its localization to telomeres.

Telomere maintenance has been implicated in cancer and ageing, and requires cooperation between a multitude of telomeric factors, including telomerase, TRF1, TRF2, RAP1, TIN2, Tankyrase, PINX1 and POT1 (refs 1-12). POT1 belongs to a family of oligonucleotide-binding (OB)-fold-containing proteins that include Oxytricha nova TEBP, Cdc13, and spPot1, which specifically recognize telomeric single-stranded DNA (ssDNA). In human cells, the loading of POT1 to telomeric ssDNA controls telomerase-mediated telomere elongation. Surprisingly, a human POT1 mutant lacking an OB fold is still recruited to telomeres. However, the exact mechanism by which this recruitment occurs remains unclear. Here we identify a novel telomere protein, PTOP, which interacts with both POT1 and TIN2. PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres. Inhibition of PTOP by RNA interference (RNAi) or disruption of the PTOP-POT1 interaction hindered the localization of POT1 to telomeres. Furthermore, expression of the respective interaction domains on PTOP and POT1 alone extended telomere length in human cells. Therefore, PTOP heterodimerizes with POT1 and regulates POT1 telomeric recruitment and telomere length.

Active Transport, Cell Nucleus↗
PubMed2004

Structural basis of membrane targeting by the Phox homology domain of cytokine-independent survival kinase (CISK-PX).

The cytokine-independent survival kinase (CISK) in the serum and glucocorticoid-regulated kinase family plays an important role in mediating cell growth and survival. N-terminal to its catalytic kinase domain, CISK contains a phox homology (PX) domain, a phosphoinositide-binding motif that directs the membrane localization of CISK and regulates CISK activity. We have determined the crystal structures of the mouse CISK-PX domain to unravel the structural basis of membrane targeting of CISK. In addition to the specific interactions conferred by the phosphoinositide-binding pocket, the structure suggests that a hydrophobic loop region and a hydrophilic beta-turn contribute to the interactions with the membrane. Furthermore, biochemical studies reveal that CISK-PX dimerizes in the presence of the linker between the PX domain and kinase domain, suggesting a multivalent mechanism in membrane localization of CISK.

Amino Acid Motifs↗
PubMed2004

The human Rap1 protein complex and modulation of telomere length.

Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells. Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2. To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis. In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex. We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2. PARP1, however, most likely interacted with hRap1 through TRF2. Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres. In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres. Deletion of a novel linker domain of hRap1 (residues 199-223), however, abolished the dominant negative effect of hRap1 overexpression. These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length.

Antigens, Nuclear↗
PubMed2004

A new XRCC1-containing complex and its role in cellular survival of methyl methanesulfonate treatment.

DNA single-strand break repair (SSBR) is important for maintaining genome stability and homeostasis. The current SSBR model derived from an in vitro-reconstituted reaction suggests that the SSBR complex mediated by X-ray repair cross-complementing protein 1 (XRCC1) is assembled sequentially at the site of damage. In this study, we provide biochemical data to demonstrate that two preformed XRCC1 protein complexes exist in cycling HeLa cells. One complex contains known enzymes that are important for SSBR, including DNA ligase 3 (DNL3), polynucleotide kinase 3'-phosphatase, and polymerase beta; the other is a new complex that contains DNL3 and the ataxia with oculomotor apraxia type 1 (AOA) gene product aprataxin. We report the characterization of the new XRCC1 complex. XRCC1 is phosphorylated in vivo and in vitro by CK2, and CK2 phosphorylation of XRCC1 on S518, T519, and T523 largely determines aprataxin binding to XRCC1 though its FHA domain. An acute loss of aprataxin by small interfering RNA renders HeLa cells sensitive to methyl methanesulfonate treatment by a mechanism of shortened half-life of XRCC1. Thus, aprataxin plays a role to maintain the steady-state protein level of XRCC1. Collectively, these data provide insights into the SSBR molecular machinery in the cell and point to the involvement of aprataxin in SSBR, thus linking SSBR to the neurological disease AOA.

Amino Acids↗
PubMed2003

An oriented peptide array library (OPAL) strategy to study protein-protein interactions.

One of the major questions in signal transduction is how the specificities of protein-protein interactions determine the assembly of distinct signaling complexes in response to stimuli. Several peptide library methods have been developed and widely used to study protein-protein interactions. These approaches primarily rely on peptide or DNA sequencing to identify the peptide or consensus motif for binding and may prove too costly or difficult to accommodate high throughput applications. We report here an oriented peptide array library (OPAL) approach that should facilitate high throughput proteomic analysis of protein-protein interactions. OPAL integrates the principles of both the oriented peptide libraries and array technologies. Hundreds of pools of oriented peptide libraries are synthesized as amino acid scan arrays. We demonstrate that these arrays can be used to map the specificities of a variety of interactions, including antibodies, protein domains such Src homology 2 domains, and protein kinases.

Animals↗
PubMed2003

Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains.

Protein phosphorylation by protein kinases may generate docking sites for other proteins. It thus allows the assembly of signaling complexes in response to kinase activation. Several protein domains that bind phosphoserine or phosphothreonine residues have been identified, including the 14-3-3, PIN1, FHA, KIX, WD-40 domain, and polo box (Yaffe, M. B., and Elia, A. E. (2001) Curr. Opin. Cell Biol. 13, 131-138; Elia, A. E., Cantley, L. C., and Yaffe, M. B. (2003) Science 299, 1228-1231). The BRCA1 COOH-terminal (BRCT) domains are protein modules found in many proteins that regulate DNA damage responses (Koonin, E. V., Altschul, S. F., and Bork, P. (1996) Nat. Genet. 13, 266-268). Whether BRCT domains can mediate phosphorylation-dependent interactions has not been systematically investigated. We report here that the BRCT domains also recognize phosphopeptides. Oriented peptide library analysis indicated that the BRCT domains from BRCA1, MDC1, BARD1, and DNA Ligase IV preferred distinct phosphoserine-containing peptides. In addition, the interaction between BRCA1 and the BRCT binding motif of BACH1 was required for BACH1 checkpoint activity. Furthermore, BRCT domains of the yeast DNA repair protein Rad9 could bind phosphopeptides, suggesting that the BRCT domains represent a class of ancient phosphopeptide-binding modules. Potential targets of BRCT domains were identified through data base search. Structural analysis of BRCA1 BRCT repeats also predicted conserved residues that may form the phosphopeptide-binding pocket. Thus, the BRCT repeats are a new family of phosphopeptide-binding domains in DNA damage responses.

Adaptor Proteins, Signal Transducing↗
PubMed2003

A Wnt- and beta -catenin-dependent pathway for mammalian cardiac myogenesis.

Acquisition of a cardiac fate by embryonic mesodermal cells is a fundamental step in heart formation. Heart development in frogs and avians requires positive signals from adjacent endoderm, including bone morphogenic proteins, and is antagonized by a second secreted signal, Wnt proteins, from neural tube. By contrast, mechanisms of mesodermal commitment to create heart muscle in mammals are largely unknown. In addition, Wnt-dependent signals can involve either a canonical beta-catenin pathway or other, alternative mediators. Here, we tested the involvement of Wnts and beta-catenin in mammalian cardiac myogenesis by using a pluripotent mouse cell line (P19CL6) that recapitulates early steps for cardiac specification. In this system, early and late cardiac genes are up-regulated by 1% DMSO, and spontaneous beating occurs. Notably, Wnt3A and Wnt8A were induced days before even the earliest cardiogenic transcription factors. DMSO induced biochemical mediators of Wnt signaling (decreased phosphorylation and increased levels of beta-catenin), which were suppressed by Frizzled-8Fc, a soluble Wnt antagonist. DMSO provoked T cell factor-dependent transcriptional activity; thus, induction of Wnt proteins by DMSO was functionally coupled. Frizzled-8Fc inhibited the induction of cardiogenic transcription factors, cardiogenic growth factors, and sarcomeric myosin heavy chains. Likewise, differentiation was blocked by constitutively active glycogen synthase kinase 3beta, an intracellular inhibitor of the Wntbeta-catenin pathway. Conversely, lithium chloride, which inhibits glycogen synthase kinase 3beta, and Wnt3A-conditioned medium up-regulated early cardiac markers and the proportion of differentiated cells. Thus, Wntbeta-catenin signaling is activated at the inception of mammalian cardiac myogenesis and is indispensable for cardiac differentiation, at least in this pluripotent model system.

Animals↗
PubMed2003

A Rac/Cdc42-specific exchange factor, GEFT, induces cell proliferation, transformation, and migration.

The Rho family of small GTPases, including Rho, Rac, and Cdc42, play essential roles in diverse cellular functions. The ability of Rho family GTPases to participate in signaling events is determined by the ratio of inactive (GDP-bound) and active (GTP-bound) forms in the cell. The activation of Rho family proteins requires the exchange of bound GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors (GEFs). The GEFs have high affinity for the guanine nucleotide-free state of the GTPases and are thought to promote GDP release by stabilizing an intermediate transition state. In this study, we have identified and characterized a new Rac/Cdc42-specific Dbl family guanine nucleotide exchange factor, named GEFT. GEFT is highly expressed in the excitable tissues, including brain, heart, and muscle. Low or very little expression was detected in other nonexcitable tissues. GEFT has specific exchange activity for Rac and Cdc42 in our in vitro GTPase exchange assays and glutathione S-transferase-PAK pull-down assays with GTP-bound Rac1 and Cdc42. Overexpression of GEFT leads to changes in cell morphology and actin cytoskeleton re-organization, including the formation of membrane microspikes, filopodia, and lamilliopodia. Furthermore, expression of GEFT in NIH3T3 cells promotes foci formation, cell proliferation, and cell migration, possibly through the activation of transcriptional factors involved in cell growth and proliferation. Together, our data suggest that GEFT is a Rac/Cdc42-specific GEF protein that regulates cell morphology, cell proliferation, and transformation.

Amino Acid Sequence↗
PubMed2002

The role of Asp-462 in regulating Akt activity.

Protein kinase Akt, an important downstream target of phosphatidylinositol 3-kinase, is one of the major survival factors in mammalian cells. It has been shown that phosphorylation of the C-terminal hydrophobic motif is required for Akt activation. The activated Akt then phosphorylates several pro-apoptotic proteins and prevents apoptosis mediated by caspases and the mitochondria. Interestingly, Akt has also been implicated to be a direct substrate of caspases in apoptotic cells induced by Fas (Widmann, C., Gibson, S., and Johnson, G. L. (1998) J. Biol. Chem. 273, 7141-7147) and anoikis (Bachelder, R. E., Wendt, M. A., Fujita, N., Tsuruo, T., and Mercurio, A. M. (2001) J. Biol. Chem. 276, 34702-34707). In this study we showed that cytokine withdrawal resulted in Akt degradation by caspases as well. Furthermore, we demonstrated residue Asp-462 of Akt1 which is just upstream of the hydrophobic motif to be the primary cleavage site. The Akt1 mutant (D462N) that prevented caspase cleavage was more stable during factor withdrawal and enhanced cell survival. The Akt truncation mutant mimicking the caspase cleavage product lost its kinase activity and functioned as a dominant negative to promote cell death. Our results suggest that the balance between Akt and caspase activity controls cell survival. In particular, caspases are able to render Akt inactive and dominantly inhibit the Akt pathway by cleaving off the C-terminal hydrophobic motif. Consequently, the survival signal is quickly down-regulated to allow apoptosis to occur.

Amino Acid Sequence↗
PubMed2002

Endosomal localization and function of sorting nexin 1.

There are 17 human members of the sorting nexin (SNX) family of proteins that contain Phox (PX) domains. Yeast orthologs function in vesicular trafficking and mammalian proteins have been implicated in endocytic trafficking of cell surface receptors. The first member of this family, SNX1, was identified via interaction with the epidermal growth factor receptor. The present studies indicate that SNX1 and SNX2 are colocalized to tubulovesicular endosomal membranes and this localization depends on PI 3-kinase activity. Point mutations in the PX domain that abolish recognition of phosphorylated phosphatidylinositol (PtdIns) in vitro abolish vesicle localization in vivo indicating that lipid binding by the PX domain is necessary for localization to vesicle membranes. Deletion of a predicted coiled-coil region in the COOH terminus of SNX1 also abolished vesicle localization, indicating that this helical domain, too, is necessary for SNX1 localization. Thus, both PX domain recognition of PtdIns and COOH terminal helical domains are necessary for localization of SNX1 with neither alone being sufficient. Regulated overexpression of the NH(2) terminus of SNX1 containing the PX domain decreased the rate of ligand-induced epidermal growth factor receptor degradation, an effect consistent with inhibition of endogenous SNX1 function in the endosome compartment. SNX1 thus functions in regulating trafficking in the endosome compartment via PX domain recognition of phosphorylated PtdIns and via interaction with other protein components.

Amino Acid Sequence↗