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2.
Microb Cell Fact ; 21(1): 52, 2022 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-35392897

RESUMO

BACKGROUND: Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked. RESULTS: A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free. CONCLUSIONS: This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.


Assuntos
Reprodutibilidade dos Testes , Animais , Cromatografia de Afinidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
4.
Oncogene ; 41(7): 960-970, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34999732

RESUMO

The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src's oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.


Assuntos
Proteômica
5.
Sci Rep ; 9(1): 19214, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844114

RESUMO

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand's cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.


Assuntos
Proteínas/genética , Proteínas/metabolismo , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dependovirus/genética , Edição de Genes/métodos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Queratinócitos/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/metabolismo
6.
Pain ; 160(10): 2305-2315, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31365468

RESUMO

Nerve growth factor (NGF) and its receptors TrkA and p75 play a key role in the development and function of peripheral nociceptive neurons. Here, we describe novel technology to selectively photoablate TrkA-positive nociceptors through delivery of a phototoxic agent coupled to an engineered NGF ligand and subsequent near-infrared illumination. We demonstrate that this approach allows for on demand and localized reversal of pain behaviors in mouse models of acute, inflammatory, neuropathic, and joint pain. To target peripheral nociceptors, we generated a SNAP-tagged NGF derivative NGF that binds to TrkA/p75 receptors but does not provoke signaling in TrkA-positive cells or elicit pain behaviors in mice. NGF was coupled to the photosensitizer IRDye700DX phthalocyanine (IR700) and injected subcutaneously. After near-infrared illumination of the injected area, behavioral responses to nociceptive mechanical and sustained thermal stimuli, but not innocuous stimuli, were substantially reduced. Similarly, in models of inflammatory, osteoarthritic, and neuropathic pain, mechanical hypersensitivity was abolished for 3 weeks after a single treatment regime. We demonstrate that this loss of pain behavior coincides with the retraction of neurons from the skin which then reinnervate the epidermis after 3 weeks corresponding with the return of mechanical hypersensitivity. Thus NGF-mediated photoablation is a minimally invasive approach to reversibly silence nociceptor input from the periphery, and control pain and hypersensitivity to mechanical stimuli.


Assuntos
Técnicas de Ablação/métodos , Fator de Crescimento Neural/administração & dosagem , Neuralgia/terapia , Nociceptores/efeitos dos fármacos , Medição da Dor/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neuralgia/fisiopatologia , Nociceptores/fisiologia , Células PC12 , Ratos
7.
Nat Biomed Eng ; 3(2): 114-125, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30944432

RESUMO

Itch-a major symptom of many chronic skin diseases-can exacerbate inflammation by provoking scratching and subsequent skin damage. Here, we show that activation, via near infrared illumination, of a phototoxic agent that selectively targets itch-sensing cells can reduce itch-associated behaviours in mice. We generated a SNAP-tagged interleukin-31 (IL-31) ligand derivative (IL-31K138A-SNAP) that selectively binds receptors on itch-associated cells, without evoking IL-31-receptor signalling or scratching, and conjugated it to the photosensitizer IRDye 700DX phthalocyanine. Subcutaneous injection of IL-31K138A-SNAP-IR700 in mice followed by near infrared illumination resulted in the long-term reversal of the scratching behaviour evoked by the pruritogenic IL-31, an effect that was associated with the selective retraction of itch-sensing neurons in the skin. We also show that a topical preparation of IL-31K138A-SNAP-IR700 reversed the behavioural and dermatological indicators of disease in mouse models of atopic dermatitis and of the genetic skin disease familial primary localized cutaneous amyloidosis. Targeted photoablation may enable itch control for the treatment of inflammatory skin diseases.


Assuntos
Comportamento Animal , Epiderme/inervação , Interleucinas/uso terapêutico , Luz , Prurido/patologia , Prurido/terapia , Células Receptoras Sensoriais/patologia , Doença Aguda , Amiloidose Familiar/patologia , Animais , Movimento Celular , Células Dendríticas/patologia , Dermatite Atópica/patologia , Dermatite Atópica/prevenção & controle , Modelos Animais de Doenças , Epiderme/patologia , Indóis/química , Queratinócitos/patologia , Camundongos Endogâmicos C57BL , Psoríase/patologia , Dermatopatias Genéticas/patologia
8.
Nat Commun ; 9(1): 1640, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29691410

RESUMO

Mechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both necessary and sufficient for producing pain from light touch after nerve injury in mice. Mice in which TrkB-Cre-expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB-positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hiperalgesia/terapia , Terapia a Laser , Glicoproteínas de Membrana/metabolismo , Neuralgia/metabolismo , Neuralgia/terapia , Proteínas Tirosina Quinases/metabolismo , Células Receptoras Sensoriais/efeitos da radiação , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Humanos , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Ligantes , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Neuralgia/genética , Neuralgia/fisiopatologia , Proteínas Tirosina Quinases/genética , Células Receptoras Sensoriais/metabolismo , Tato/efeitos da radiação
9.
Structure ; 23(5): 893-902, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25914053

RESUMO

Regulation of c-Src activity by the intrinsically disordered Unique domain has recently been demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains.


Assuntos
Lipídeos/química , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Quinases da Família src/química , Quinases da Família src/metabolismo , Sítios de Ligação , Proteína Tirosina Quinase CSK , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Domínios de Homologia de src
10.
Sci Rep ; 3: 1295, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23416516

RESUMO

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.


Assuntos
Lipídeos/química , Quinases da Família src/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Proteína Tirosina Quinase CSK , Calmodulina/química , Calmodulina/metabolismo , Galinhas , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Oócitos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Fosforilação , Ligação Proteica , Xenopus/crescimento & desenvolvimento , Domínios de Homologia de src , Quinases da Família src/química , Quinases da Família src/genética
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