O-linked β-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including... more O-linked β-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including glucose and glutamine; its dysregulation leads to molecular and pathological alterations that cause diseases. Here we report that O-GlcNAc directly regulates de novo nucleotide synthesis and nicotinamide adenine dinucleotide (NAD) production upon abnormal metabolic states. Phosphoribosyl pyrophosphate synthetase 1 (PRPS1), the key enzyme of the de novo nucleotide synthesis pathway, is O-GlcNAcylated by O-GlcNAc transferase (OGT), which triggers PRPS1 hexamer formation and relieves nucleotide product-mediated feedback inhibition, thereby boosting PRPS1 activity. PRPS1 O-GlcNAcylation blocked AMPK binding and inhibited AMPK-mediated PRPS1 phosphorylation. OGT still regulates PRPS1 activity in AMPK-deficient cells. Elevated PRPS1 O-GlcNAcylation promotes tumorigenesis and confers resistance to chemoradiotherapy in lung cancer. Furthermore, Arts-syndrome-associated PRPS1 R196W mutant exhibi...
Integrins are ubiquitously expressed cell-adhesion proteins. Talin is required for integrin activ... more Integrins are ubiquitously expressed cell-adhesion proteins. Talin is required for integrin activation through an inside-out signaling pathway, during which talin is recruited to the plasma membrane (PM) by RAP1 directly or through its effector, RAP1-Interacting Adaptor Molecule (RIAM). RIAM also activates talin from autoinhibition by binding to talin head domain. A helical talin-binding segment (TBS) in RIAM mediates both talin activation and recruitment by binding to two distinct sites in talin head and rod domains respectively. The bi-specificity of the TBS fragment allows us to develop a new strategy to suppress talin-induced integrin activation through a "double-hit" approach. We designed an experimental peptidomimetic inhibitor by engineering a hydrocarbon "staple" in the helical TBS fragment to mask the integrin binding site in the talin head. The stapled peptide (S-TBS) exhibits a stronger binding affinity with talin and inhibits talin:integrin interactio...
Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound tali... more Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the β integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+. Here we show that kindlin-1 can replace Mn2+ to mediate β3 integrin clustering induced by the talin head, but not that induced by the F2–F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for β3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and β3-integrins, in order to activate the β3 integrin heterodimer, further detailing the mechanism by which the talin–kin...
Cancer immunotherapy has accomplished significant progresses on treatment of various cancers in t... more Cancer immunotherapy has accomplished significant progresses on treatment of various cancers in the past decade; however, recent studies revealed more and more heterogeneity in tumor microenvironment which cause unneglectable therapy resistance. A central phenomenon in tumor malignancy is metabolic dysfunctionality; it reprograms metabolic homeostasis in tumor and stromal cells thus affecting metabolic modifications on specific proteins. These posttranslational modifications include glycosylation and palmitoylation, which usually alter the protein localization, stability, and function. Many of these proteins participate in acute or chronic inflammation and play critical roles in tumorigenesis and progression. Therefore, targeting these metabolic modifications in immune checkpoints and inflammation provides an attractive therapeutic strategy for certain cancers. In this review, we summarize the recent progresses on metabolic modifications in this field, focus on the mechanisms on how...
Proceedings of the National Academy of Sciences, 2020
Significance Although efforts have been made to determine the structure of talin and the way it i... more Significance Although efforts have been made to determine the structure of talin and the way it interacts with integrins through the “head” domain, our work shows now that many of the previous mechanistic models based on the talin adapter are likely to be misleading as they are constructed on a crystal structure representing an improperly folded talin head domain. In this work, we identified the problem with the current talin head model and proposed a FERM-folded talin head. By analyzing these structural features of the FERM-folded talin head in a cellular context, involving also the kindlin adapter, we are making a critical and unprecedented contribution to the understanding and regulation of cell-matrix adhesions.
The HSP70 family of molecular chaperones function to maintain protein quality control and homeost... more The HSP70 family of molecular chaperones function to maintain protein quality control and homeostasis. The major stress-induced form, HSP70 (also called HSP72 or HSPA1A) is considered an important anti-cancer drug target because it is constitutively overexpressed in a number of human cancers and promotes cancer cell survival. All HSP70 family members contain two functional domains: an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate-binding domain (SBD); the latter is subdivided into SBDα and SBDβ subdomains. The NBD and SBD structures of the bacterial ortholog, DnaK, have been characterized, but only the isolated NBD and SBDα segments of eukaryotic HSP70 proteins have been determined. Here we report the crystal structure of the substrate-bound human HSP70-SBD to 2 angstrom resolution. The overall fold of this SBD is similar to the corresponding domain in the substrate-bound DnaK structures, confirming a similar overall architecture of the orthologous ba...
O-linked β-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including... more O-linked β-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including glucose and glutamine; its dysregulation leads to molecular and pathological alterations that cause diseases. Here we report that O-GlcNAc directly regulates de novo nucleotide synthesis and nicotinamide adenine dinucleotide (NAD) production upon abnormal metabolic states. Phosphoribosyl pyrophosphate synthetase 1 (PRPS1), the key enzyme of the de novo nucleotide synthesis pathway, is O-GlcNAcylated by O-GlcNAc transferase (OGT), which triggers PRPS1 hexamer formation and relieves nucleotide product-mediated feedback inhibition, thereby boosting PRPS1 activity. PRPS1 O-GlcNAcylation blocked AMPK binding and inhibited AMPK-mediated PRPS1 phosphorylation. OGT still regulates PRPS1 activity in AMPK-deficient cells. Elevated PRPS1 O-GlcNAcylation promotes tumorigenesis and confers resistance to chemoradiotherapy in lung cancer. Furthermore, Arts-syndrome-associated PRPS1 R196W mutant exhibi...
Integrins are ubiquitously expressed cell-adhesion proteins. Talin is required for integrin activ... more Integrins are ubiquitously expressed cell-adhesion proteins. Talin is required for integrin activation through an inside-out signaling pathway, during which talin is recruited to the plasma membrane (PM) by RAP1 directly or through its effector, RAP1-Interacting Adaptor Molecule (RIAM). RIAM also activates talin from autoinhibition by binding to talin head domain. A helical talin-binding segment (TBS) in RIAM mediates both talin activation and recruitment by binding to two distinct sites in talin head and rod domains respectively. The bi-specificity of the TBS fragment allows us to develop a new strategy to suppress talin-induced integrin activation through a "double-hit" approach. We designed an experimental peptidomimetic inhibitor by engineering a hydrocarbon "staple" in the helical TBS fragment to mask the integrin binding site in the talin head. The stapled peptide (S-TBS) exhibits a stronger binding affinity with talin and inhibits talin:integrin interactio...
Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound tali... more Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the β integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+. Here we show that kindlin-1 can replace Mn2+ to mediate β3 integrin clustering induced by the talin head, but not that induced by the F2–F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for β3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and β3-integrins, in order to activate the β3 integrin heterodimer, further detailing the mechanism by which the talin–kin...
Cancer immunotherapy has accomplished significant progresses on treatment of various cancers in t... more Cancer immunotherapy has accomplished significant progresses on treatment of various cancers in the past decade; however, recent studies revealed more and more heterogeneity in tumor microenvironment which cause unneglectable therapy resistance. A central phenomenon in tumor malignancy is metabolic dysfunctionality; it reprograms metabolic homeostasis in tumor and stromal cells thus affecting metabolic modifications on specific proteins. These posttranslational modifications include glycosylation and palmitoylation, which usually alter the protein localization, stability, and function. Many of these proteins participate in acute or chronic inflammation and play critical roles in tumorigenesis and progression. Therefore, targeting these metabolic modifications in immune checkpoints and inflammation provides an attractive therapeutic strategy for certain cancers. In this review, we summarize the recent progresses on metabolic modifications in this field, focus on the mechanisms on how...
Proceedings of the National Academy of Sciences, 2020
Significance Although efforts have been made to determine the structure of talin and the way it i... more Significance Although efforts have been made to determine the structure of talin and the way it interacts with integrins through the “head” domain, our work shows now that many of the previous mechanistic models based on the talin adapter are likely to be misleading as they are constructed on a crystal structure representing an improperly folded talin head domain. In this work, we identified the problem with the current talin head model and proposed a FERM-folded talin head. By analyzing these structural features of the FERM-folded talin head in a cellular context, involving also the kindlin adapter, we are making a critical and unprecedented contribution to the understanding and regulation of cell-matrix adhesions.
The HSP70 family of molecular chaperones function to maintain protein quality control and homeost... more The HSP70 family of molecular chaperones function to maintain protein quality control and homeostasis. The major stress-induced form, HSP70 (also called HSP72 or HSPA1A) is considered an important anti-cancer drug target because it is constitutively overexpressed in a number of human cancers and promotes cancer cell survival. All HSP70 family members contain two functional domains: an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate-binding domain (SBD); the latter is subdivided into SBDα and SBDβ subdomains. The NBD and SBD structures of the bacterial ortholog, DnaK, have been characterized, but only the isolated NBD and SBDα segments of eukaryotic HSP70 proteins have been determined. Here we report the crystal structure of the substrate-bound human HSP70-SBD to 2 angstrom resolution. The overall fold of this SBD is similar to the corresponding domain in the substrate-bound DnaK structures, confirming a similar overall architecture of the orthologous ba...
Uploads
Papers by Pingfeng Zhang