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1.
Proc Natl Acad Sci U S A ; 118(32)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34349020

RESUMO

Family with sequence similarity 20C (Fam20C), the major protein kinase in the secretory pathway, generates the vast majority of the secreted phosphoproteome. However, the regulatory mechanisms of Fam20C transport, secretion, and function remain largely unexplored. Here, we show that Fam20C exists as a type II transmembrane protein within the secretory compartments, with its N-terminal signal peptide-like region serving as a membrane anchor for Golgi retention. The secretion and kinase activity of Fam20C are governed by site-1 protease (S1P), a key regulator of cholesterol homeostasis. We find that only mature Fam20C processed by S1P functions in osteoblast differentiation and mineralization. Together, our findings reveal a unique mechanism for Fam20C secretion and activation via proteolytic regulation, providing a molecular link between biomineralization and lipid metabolism.

2.
Nat Commun ; 12(1): 5000, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404805

RESUMO

The successive emergences and accelerating spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages and evolved resistance to some ongoing clinical therapeutics increase the risks associated with the coronavirus disease 2019 (COVID-19) pandemic. An urgent intervention for broadly effective therapies to limit the morbidity and mortality of COVID-19 and future transmission events from SARS-related coronaviruses (SARSr-CoVs) is needed. Here, we isolate and humanize an angiotensin-converting enzyme-2 (ACE2)-blocking monoclonal antibody (MAb), named h11B11, which exhibits potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages. When administered therapeutically or prophylactically in the hACE2 mouse model, h11B11 alleviates and prevents SARS-CoV-2 replication and virus-induced pathological syndromes. No significant changes in blood pressure and hematology chemistry toxicology were observed after injections of multiple high dosages of h11B11 in cynomolgus monkeys. Analysis of the structures of the h11B11/ACE2 and receptor-binding domain (RBD)/ACE2 complexes shows hindrance and epitope competition of the MAb and RBD for the receptor. Together, these results suggest h11B11 as a potential therapeutic countermeasure against SARS-CoV, SARS-CoV-2, and escape variants.


Assuntos
Enzima de Conversão de Angiotensina 2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/administração & dosagem , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/mortalidade , COVID-19/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Epitopos , Feminino , Células HEK293 , Haplorrinos , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pandemias , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Células Vero , Ativação Viral
4.
Sci China Life Sci ; 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34431042

RESUMO

The outbreak of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has created a global health crisis. SARS-CoV-2 infects varieties of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative viral entry pathways. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. Beyond known host proteins, i.e., ACE2, TMPRSS2, and NRP1, we identified multiple host components, among which LDLRAD3, TMEM30A, and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike's N-terminal domain (NTD). Their essential and physiological roles have been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in an ACE2-independent fashion. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of COVID-19 countermeasures.

5.
Cell Res ; 31(7): 732-741, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34021265

RESUMO

SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242-244 deletion (242-244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242-244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Imunidade Humoral , SARS-CoV-2/imunologia , Vacinas de Produtos Inativados/farmacologia , Animais , Anticorpos Neutralizantes/sangue , COVID-19/sangue , Vacinas contra COVID-19/imunologia , Linhagem Celular , Células HEK293 , Humanos , Modelos Moleculares , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/farmacologia
6.
Oncogene ; 40(11): 1942-1956, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33603165

RESUMO

Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptors tyrosine kinases can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase nonreceptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anticancer therapies.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/genética , Processamento de Proteína Pós-Traducional/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Acetilação/efeitos dos fármacos , Animais , Benzodioxóis/farmacologia , Membrana Celular/genética , Citoplasma/genética , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutação/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Quinazolinas/farmacologia , Tirosina/genética
7.
Cell ; 183(4): 1013-1023.e13, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32970990

RESUMO

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Reações Antígeno-Anticorpo , Sítios de Ligação , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cricetinae , Microscopia Crioeletrônica , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Feminino , Pulmão/patologia , Masculino , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Estrutura Quaternária de Proteína , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
8.
Elife ; 92020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32975514

RESUMO

Matriglycan [-GlcA-ß1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-ß1,3-GlcNAc-ß1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.


Assuntos
Distroglicanas/metabolismo , Expressão Gênica , Músculo Esquelético/fisiologia , N-Acetilglucosaminiltransferases/genética , Proteínas Quinases/genética , Animais , Masculino , Manose/química , Camundongos , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo
9.
Angew Chem Int Ed Engl ; 59(37): 16122-16127, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32506656

RESUMO

Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post-translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS-PAGE, LC-MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X-ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein-topology engineering.


Assuntos
Antracenos/química , Cristalografia por Raios X/métodos , Antracenos/síntese química , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Estrutura Molecular , Proteólise
10.
Cell Res ; 30(7): 602-609, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32398862

RESUMO

Secretory Immunoglobulin A (SIgA) is the most abundant antibody at the mucosal surface. It possesses two additional subunits besides IgA: the joining chain (J-chain) and secretory component (SC). SC is the ectodomain of the polymeric immunoglobulin receptor (pIgR), which functions to transport IgA to the mucosa. How the J-chain and pIgR/SC facilitate the assembly and secretion of SIgA remains incompletely understood. Furthermore, during the infection of Streptococcus pneumoniae, the pneumococcal adhesin SpsA hijacks pIgR/SC and SIgA to gain entry to human cells and evade host defense. How SpsA targets pIgR/SC and SIgA also remains elusive. Here we report a cryo-electron microscopy structure of the Fc region of IgA1 (Fcα) in complex with the J-chain and SC (Fcα-J-SC), which reveals the organization principle of SIgA. We also present a structure of Fcα-J-SC complexed with SpsA, which uncovers the specific interactions between SpsA and human pIgR/SC. These results advance the molecular understanding of SIgA and shed light on S. pneumoniae pathogenesis.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Imunoglobulina A Secretora/química , Imunoglobulina A Secretora/metabolismo , Adesinas Bacterianas/química , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Humanos , Imunoglobulina A Secretora/ultraestrutura , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Multimerização Proteica
11.
Cell ; 182(1): 73-84.e16, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32425270

RESUMO

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Linfócitos B/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Análise de Célula Única , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , COVID-19 , Convalescença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Pandemias , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Éxons VDJ
12.
Science ; 367(6481): 1014-1017, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32029689

RESUMO

Immunoglobulin M (IgM) plays a pivotal role in both humoral and mucosal immunity. Its assembly and transport depend on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR), but the underlying molecular mechanisms of these processes are unclear. We report a cryo-electron microscopy structure of the Fc region of human IgM in complex with the J-chain and pIgR ectodomain. The IgM-Fc pentamer is formed asymmetrically, resembling a hexagon with a missing triangle. The tailpieces of IgM-Fc pack into an amyloid-like structure to stabilize the pentamer. The J-chain caps the tailpiece assembly and bridges the interaction between IgM-Fc and the polymeric immunoglobulin receptor, which undergoes a large conformational change to engage the IgM-J complex. These results provide a structural basis for the function of IgM.


Assuntos
Imunoglobulina M/química , Imunoglobulina M/imunologia , Receptores de Imunoglobulina Polimérica/química , Microscopia Crioeletrônica , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Cadeias J de Imunoglobulina/química , Cadeias J de Imunoglobulina/imunologia , Conformação Proteica , Multimerização Proteica
13.
Proc Natl Acad Sci U S A ; 116(49): 24881-24891, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31754034

RESUMO

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.


Assuntos
Bortezomib/farmacologia , Processos Neoplásicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , TYK2 Quinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Linhagem Celular Tumoral , Feminino , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mieloma Múltiplo , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Neoplasias de Mama Triplo Negativas/patologia
14.
Curr Biol ; 29(19): 3256-3265.e5, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31564495

RESUMO

In angiosperms, two sperm cells are transported and delivered by the pollen tube to the ovule to achieve double fertilization. Extensive communication takes place between the pollen tube and the female tissues until the sperm cell cargo is ultimately released. During this process, a pollen tube surface-located receptor complex composed of ANXUR1/2 (ANX1/2) and Buddha's Paper Seal 1/2 (BUPS1/2) was reported to control the maintenance of pollen tube integrity by perceiving the autocrine peptide ligands rapid alkalinization factor 4 and 19 (RALF4/19). It was further hypothesized that pollen-tube rupture to release sperm is caused by the paracrine RALF34 peptide from the ovule interfering with this signaling pathway. In this study, we identified two Arabidopsis pollen-tube-expressed glycosylphosphatidylinositol-anchored proteins (GPI-APs), LORELEI-like-GPI-anchored protein 2 (LLG2) and LLG3, as co-receptors in the BUPS-ANX receptor complex. llg2 llg3 double mutants exhibit severe fertility defects. Mutant pollen tubes rupture early during the pollination process. Furthermore, LLG2 and LLG3 interact with ectodomains of both BUPSs and ANXURs, and this interaction is remarkably enhanced by the presence of RALF4/19 peptides. We further demonstrate that the N terminus (including a YISY motif) of the RALF4 peptide ligand interacts strongly with BUPS-ANX receptors but weakly with LLGs and is essential for its biological function, and its C-terminal region is sufficient for LLG binding. In conclusion, we propose that LLG2/3 serve as co-receptors during BUPS/ANX-RALF signaling and thereby further establish the importance of GPI-APs as key regulators in plant reproduction processes.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Ligadas por GPI/genética , Tubo Polínico/crescimento & desenvolvimento , Transdução de Sinais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Ligadas por GPI/metabolismo , Ligantes
15.
PLoS Pathog ; 15(6): e1007876, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31216343

RESUMO

The guanylate-binding proteins (GBPs) belong to the dynamin superfamily of GTPases and function in cell-autonomous defense against intracellular pathogens. IpaH9.8, an E3 ligase from the pathogenic bacterium Shigella flexneri, ubiquitinates a subset of GBPs and leads to their proteasomal degradation. Here we report the structure of a C-terminally truncated GBP1 in complex with the IpaH9.8 Leucine-rich repeat (LRR) domain. IpaH9.8LRR engages the GTPase domain of GBP1, and differences in the Switch II and α3 helix regions render some GBPs such as GBP3 and GBP7 resistant to IpaH9.8. Comparisons with other IpaH structures uncover interaction hot spots in their LRR domains. The C-terminal region of GBP1 undergoes a large rotation compared to previously determined structures. We further show that the C-terminal farnesylation modification also plays a role in regulating GBP1 conformation. Our results suggest a general mechanism by which the IpaH proteins target their cellular substrates and shed light on the structural dynamics of the GBPs.


Assuntos
Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Shigella flexneri/enzimologia , Ubiquitina-Proteína Ligases/química , Proteínas de Bactérias/genética , Domínios Proteicos , Shigella flexneri/genética , Ubiquitina-Proteína Ligases/genética
16.
Science ; 364(6443)2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31147494

RESUMO

Reproductive isolation is a prerequisite for speciation. Failure of communication between female tissues of the pistil and paternal pollen tubes imposes hybridization barriers in flowering plants. Arabidopsis thaliana LURE1 (AtLURE1) peptides and their male receptor PRK6 aid attraction of the growing pollen tube to the ovule. Here, we report that the knockout of the entire AtLURE1 gene family did not affect fertility, indicating that AtLURE1-PRK6-mediated signaling is not required for successful fertilization within one Arabidopsis species. AtLURE1s instead function as pollen tube emergence accelerators that favor conspecific pollen over pollen from other species and thus promote reproductive isolation. We also identified maternal peptides XIUQIU1 to -4, which attract pollen tubes regardless of species. Cooperation between ovule attraction and pollen tube growth acceleration favors conspecific fertilization and promotes reproductive isolation.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Genes de Plantas/fisiologia , Peptídeos/fisiologia , Tubo Polínico/crescimento & desenvolvimento , Isolamento Reprodutivo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Técnicas de Inativação de Genes , Peptídeos/genética , Tubo Polínico/genética
17.
Nat Chem Biol ; 15(3): 213-216, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30617292

RESUMO

The identification of host protein substrates is key to understanding effector glycosyltransferases secreted by pathogenic bacteria and to using them for glycoprotein engineering. Here we report a chemical method for tagging, enrichment, and site-specific proteomic profiling of effector-modified proteins in host cells. Using this method, we discover that Legionella effector SetA α-O-glucosylates various eukaryotic proteins by recognizing a S/T-X-L-P/G sequence motif, which can be exploited to site-specifically introduce O-glucose on recombinant proteins.


Assuntos
Glicosiltransferases/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteínas de Bactérias , Eucariotos , Glucosiltransferases/metabolismo , Interações Hospedeiro-Patógeno , Legionella/metabolismo , Proteômica , Proteínas Recombinantes
18.
Proc Natl Acad Sci U S A ; 115(32): 8155-8160, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-29987021

RESUMO

Curcumin, the active ingredient in Curcuma longa, has been in medicinal use since ancient times. However, the therapeutic targets and signaling cascades modulated by curcumin have been enigmatic despite extensive research. Here we identify dual-specificity tyrosine-regulated kinase 2 (DYRK2), a positive regulator of the 26S proteasome, as a direct target of curcumin. Curcumin occupies the ATP-binding pocket of DYRK2 in the cocrystal structure, and it potently and specifically inhibits DYRK2 over 139 other kinases tested in vitro. As a result, curcumin diminishes DYRK2-mediated 26S proteasome phosphorylation in cells, leading to reduced proteasome activity and impaired cell proliferation. Interestingly, curcumin synergizes with the therapeutic proteasome inhibitor carfilzomib to induce apoptosis in a variety of proteasome-addicted cancer cells, while this drug combination exhibits modest to no cytotoxicity to noncancerous cells. In a breast cancer xenograft model, curcumin treatment significantly reduces tumor burden in immunocompromised mice, showing a similar antitumor effect as CRISPR/Cas9-mediated DYRK2 depletion. These results reveal an unexpected role of curcumin in DYRK2-proteasome inhibition and provide a proof-of-concept that pharmacological manipulation of proteasome regulators may offer new opportunities for anticancer treatment.


Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Curcumina/uso terapêutico , Sinergismo Farmacológico , Feminino , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Células HEK293 , Humanos , Concentração Inibidora 50 , Camundongos , Neoplasias/patologia , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
EMBO J ; 37(14)2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29858230

RESUMO

Family with sequence similarity 20C (Fam20C), the physiological Golgi casein kinase, phosphorylates numerous secreted proteins that are involved in a wide variety of biological processes. However, the role of Fam20C in regulating proteins in the endoplasmic reticulum (ER) lumen is largely unknown. Here, we report that Fam20C interacts with various luminal proteins and that its depletion results in a more reduced ER lumen. We further show that ER oxidoreductin 1α (Ero1α), the pivotal sulfhydryl oxidase that catalyzes disulfide formation in the ER, is phosphorylated by Fam20C in the Golgi apparatus and retrograde-transported to the ER mediated by ERp44. The phosphorylation of Ser145 greatly enhances Ero1α oxidase activity and is critical for maintaining ER redox homeostasis and promoting oxidative protein folding. Notably, phosphorylation of Ero1α is induced under hypoxia, reductive stress, and secretion-demanding conditions such as mammalian lactation. Collectively, our findings open a door to uncover how oxidative protein folding is regulated by phosphorylation in the secretory pathway.


Assuntos
Caseína Quinase I/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Processamento de Proteína Pós-Traducional , Células HeLa , Células Hep G2 , Humanos , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Oxirredução , Fosforilação , Transporte Proteico
20.
Proc Natl Acad Sci U S A ; 115(27): 7039-7044, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29915090

RESUMO

The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid ß-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2ß2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. Deletion of a helical hairpin in TFPß decreases its binding to the liposomes in vitro and reduces its membrane targeting in cells. Our results provide the structural basis for TFP function and have important implications for fatty acid oxidation related diseases.


Assuntos
Microscopia Crioeletrônica , Proteína Mitocondrial Trifuncional/ultraestrutura , Humanos , Proteína Mitocondrial Trifuncional/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
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