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Sarcopenia is a complex age-associated syndrome of progressive loss of muscle mass and strength. Although this condition is influenced by many factors, age-related changes in immune function including immune cell dynamics, and chronic inflammation contribute to its progression. The complex interplay between the immune system, gut-muscle axis, and autophagy further underscores their important roles in sarcopenia pathogenesis. Immunomodulation has emerged as a promising strategy to counteract sarcopenia. Traditional management approaches to treat sarcopenia including physical exercise and nutritional supplementation, and the emerging technologies of biophysical stimulation demonstrated the importance of immunomodulation and regulation of macrophages and T cells and reduction of chronic inflammation. Treatments to alleviate low-grade inflammation in older adults by modulating gut microbial composition and diversity further combat sarcopenia. Furthermore, some pharmacological interventions, nano-medicine, and cell therapies targeting muscle, gut microbiota, or autophagy present additional avenues for immunomodulation in sarcopenia. This narrative review explores the immunological underpinnings of sarcopenia, elucidating the relationship between the immune system and muscle during ageing. Additionally, the review discusses new areas such as the gut-muscle axis and autophagy, which bridge immune system function and muscle health. Insights into current and potential approaches for sarcopenia management through modulation of the immune system are provided, along with suggestions for future research directions and therapeutic strategies. We aim to guide further investigation into clinical immunological biomarkers and identify indicators for sarcopenia diagnosis and potential treatment targets to combat this condition. We also aim to draw attention to the importance of considering immunomodulation in the clinical management of sarcopenia.
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Macroautophagy/autophagy is a highly conserved process for the degradation of cellular components and plays an essential role in cellular homeostasis maintenance. During autophagy, specialized double-membrane vesicles known as autophagosomes are formed and sequester cytoplasmic cargoes and deliver them to lysosomes or vacuoles for breakdown. Central to this process are autophagy-related (ATG) proteins, with the ATG9-the only integral membrane protein in this core machinery-playing a central role in mediating autophagosome formation. Recent years have witnessed the maturation of cryo-electron microscopy (cryo-EM) and single-particle analysis into powerful tools for high-resolution structural determination of protein complexes. These advancements have significantly deepened our understanding of the intricate molecular mechanisms underlying autophagosome biogenesis. In this study, we present a protocol detailing the acquisition of the three-dimensional structure of ATG9 from Arabidopsis thaliana. The structural resolution achieved 7.8 Å determined by single-particle cryo-electron microscopy (cryo-EM).
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Proteínas de Arabidopsis , Arabidopsis , Proteínas Relacionadas con la Autofagia , Microscopía por Crioelectrón , Microscopía por Crioelectrón/métodos , Arabidopsis/metabolismo , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestructura , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/química , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Autofagia , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructuraRESUMEN
Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.
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Fitosteroles , Esteroles , Esteroles/metabolismo , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Fitosteroles/metabolismoRESUMEN
Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.
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Neuropilina-2 , Neoplasias de la Mama Triple Negativas , Humanos , Neuropilina-2/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Unión Proteica , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Neuropilina-1/metabolismoRESUMEN
Bacterial acquisition of metabolites is largely facilitated by transporters with unique substrate scopes. The tripartite ATP-independent periplasmic (TRAP) transporters comprise a large family of bacterial proteins that facilitate the uptake of a variety of small molecules. It has been reported that some TRAP systems encode a fourth protein, the T component. The T-component, or TatT, is predicted to be a periplasmic-facing lipoprotein that enables the uptake of metabolites from the outer membrane. However, no substrates were revealed for any TatT and their functional role(s) remained enigmatic. We recently identified a homolog in Methylococcus capsulatus that binds to sterols, and herein, we report two additional homologs that demonstrate a preference for long-chain fatty acids. Our bioinformatics, quantitative analyses of protein-ligand interactions, and high-resolution crystal structures suggest that TatTs might facilitate the trafficking of hydrophobic or lipophilic substrates and represent a new class of bacterial lipid and fatty acid transporters.
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Bacterias , Proteínas de Transporte de Membrana , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Ácidos Grasos/metabolismoRESUMEN
Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportunities to probe protein function and validate therapeutic targets. Here, we describe the optimization of nanobodies specific for BCL11A, a validated target for the treatment of hemoglobin disorders. We obtained first-generation nanobodies directed to a region of BCL11A comprising zinc fingers 4 to 6 (ZF456) from a synthetic yeast surface display library, and employed error-prone mutagenesis, structural determination, and molecular modeling to enhance binding affinity. Engineered nanobodies recognized ZF6 and mediated targeted protein degradation (TPD) of BCL11A protein in erythroid cells, leading to the anticipated reactivation of fetal hemoglobin (HbF) expression. Evolved nanobodies distinguished BCL11A from its close paralog BCL11B, which shares an identical DNA-binding specificity. Given the ease of manipulation of nanobodies and their exquisite specificity, nanobody-mediated TPD of TFs should be suitable for dissecting regulatory relationships of TFs and gene targets and validating therapeutic potential of proteins of interest.
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Anticuerpos de Dominio Único , Humanos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Hemoglobina Fetal/metabolismoRESUMEN
The human VPS10 domain-containing receptor SorCS3 belongs to the Vps10p-domain receptor family and is an important receptor for regulating normal cellular functions via protein sorting. Here, we determined the cryo-EM structure of the full-length SorCS3 receptor and further found that there were at least three distinct conformations (monomer, M-shaped dimer and N-shaped dimer) of SorCS3 in the apo state. The differences between the two dimer conformations were caused by PKD1-2 assembly. In contrast to its homologous proteins, the conserved residues GLN198, ARG678, TYR430, GLU1020 and ASP1024 may be key points for its dimerization and for protein/polypeptide binding. These results showed the structural details of apo-SorCS3, which provides a foundation for elucidating the mechanism of protein sorting.
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Proteínas Portadoras , Proteínas del Tejido Nervioso , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Humanos , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Transporte de Proteínas , Receptores de Superficie Celular/metabolismoRESUMEN
Type 1 insulin-like growth factor receptor (IGF1R) plays an important role in regulating cellular metabolism and cell growth and has been identified as an anticancer drug target. Although previous studies have revealed some structures of IGF1R with different ligands, the continuous dynamic conformation change remains unclear. Here, we report 10 distinct structures (7.9-3.6 Å) of IGF1R bound to IGF1 or insulin to reveal the polymorphic conformations of ligand-bound IGF1R. These results showed that the α-CT2, disulfide bond (C670-C670'), and FnIII-2 domains had the most flexible orientations for the conformational change that occurs when ligands bind to the receptor. In addition, we found one special conformation (tentatively named the diverter-switch state) in both complexes, which may be one of the apo-IGF1R forms under ligand-treatment conditions. Hence, these results illustrated the mechanism of how different ligands could bind to human IGF1R and provided a rational template for drug design.
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Insulina , Receptor IGF Tipo 1 , Proliferación Celular , Microscopía por Crioelectrón , Humanos , Insulina/metabolismo , Ligandos , Dominios Proteicos , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismoRESUMEN
Sorting-related receptor with A-type repeats (SORLA) is an important receptor for regulating normal cellular functions via protein sorting. Here, we determined the structures of the full-length SORLA and identified two distinct conformations of apo-SORLA using single-particle cryogenic electron microscopy. In contrast to homologous proteins, both monomer and dimer forms of SORLA existed in a neutral solution. Only three hydrogen bonds in the vicinity of the dimer interface implied the involvement in dimerization. The orientation of residue R490 was a key point for ligand binding. These results suggest a unique mechanism of SORLA dimerization for protein trafficking.
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Proteínas Adaptadoras del Transporte Vesicular , Proteínas Relacionadas con Receptor de LDL , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Proteínas Relacionadas con Receptor de LDL/metabolismo , Transporte de ProteínasRESUMEN
Proximity-based strategies to degrade proteins have enormous therapeutic potential in medicine, but the technologies are limited to proteins for which small molecule ligands exist. The identification of such ligands for therapeutically relevant but "undruggable" proteins remains challenging. Herein, we employed yeast surface display of synthetic nanobodies to identify a protein ligand selective for BCL11A, a critical repressor of fetal globin gene transcription. Fusion of the nanobody to a cell-permeant miniature protein and an E3 adaptor creates a degrader that depletes cellular BCL11A in differentiated primary erythroid precursor cells, thereby inducing the expression of fetal hemoglobin, a modifier of clinical severity of sickle cell disease and ß-thalassemia. Our strategy provides a means of fetal hemoglobin induction through reversible, temporal modulation of BCL11A. Additionally, it establishes a new paradigm for the targeted degradation of previously intractable proteins.
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The yeast protein Rad5 and its orthologs in other eukaryotes promote replication stress tolerance and cell survival using their multiple activities, including ubiquitin ligase, replication fork remodeling and DNA lesion targeting activities. Here, we present the crystal structure of a nearly full-length Rad5 protein. The structure shows three distinct, but well-connected, domains required for Rad5's activities. The spatial arrangement of these domains suggest that different domains can have autonomous activities but also undergo intrinsic coordination. Moreover, our structural, biochemical and cellular studies demonstrate that Rad5's HIRAN domain mediates interactions with the DNA metabolism maestro factor PCNA and contributes to its poly-ubiquitination, binds to DNA and contributes to the Rad5-catalyzed replication fork regression, defining a new type of HIRAN domains with multiple activities. Our work provides a framework to understand how Rad5 integrates its various activities in replication stress tolerance.
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Adaptación Fisiológica , Proteínas Fúngicas/metabolismo , Kluyveromyces/metabolismo , Estrés Fisiológico , Biocatálisis , Secuencia Conservada , ADN/metabolismo , Proteínas Fúngicas/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
Aminoacyl-tRNA synthetases (ARSs) function to transfer amino acids to cognate tRNA molecules, which are required for protein translation. To date, biallelic mutations in 31 ARS genes are known to cause recessive, early-onset severe multi-organ diseases. VARS encodes the only known valine cytoplasmic-localized aminoacyl-tRNA synthetase. Here, we report seven patients from five unrelated families with five different biallelic missense variants in VARS. Subjects present with a range of global developmental delay, epileptic encephalopathy and primary or progressive microcephaly. Longitudinal assessment demonstrates progressive cortical atrophy and white matter volume loss. Variants map to the VARS tRNA binding domain and adjacent to the anticodon domain, and disrupt highly conserved residues. Patient primary cells show intact VARS protein but reduced enzymatic activity, suggesting partial loss of function. The implication of VARS in pediatric neurodegeneration broadens the spectrum of human diseases due to mutations in tRNA synthetase genes.
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Epilepsia/genética , Mutación , Valina-ARNt Ligasa/genética , Alelos , Anticodón , Niño , Preescolar , Progresión de la Enfermedad , Epilepsia/enzimología , Epilepsia/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Estudios Longitudinales , Mutación con Pérdida de Función , Masculino , Microcefalia/enzimología , Microcefalia/genética , Modelos Moleculares , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Linaje , Biosíntesis de Proteínas , Dominios y Motivos de Interacción de Proteínas , ARN de Transferencia/genética , Secuenciación del Exoma , Secuenciación Completa del GenomaRESUMEN
The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.
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Aminoacil-ARNt Sintetasas/genética , Enfermedades Pulmonares/genética , Mutación/genética , Adolescente , Alelos , Enfermedad de Charcot-Marie-Tooth/genética , Preescolar , Femenino , Genes Recesivos/genética , Heterocigoto , Humanos , Lactante , Masculino , Biosíntesis de Proteínas/genéticaRESUMEN
The cAMP response element (CRE) binding protein (CREB) is central in the transcription regulation by cAMP, and the CREB-regulated transcriptional coactivators (CRTCs) play critical roles in CREB-mediated transcription activation. Upon stimulation, CRTCs translocate into the nucleus and complex with CREB on CRE promoters to activate target gene transcription. Their physiological importance is underscored by their function in energy balance, long-term memory, longevity and other processes. The CREB binding domain on CRTCs has been mapped, which interacts with the CREB basic leucine zipper domain that also mediates interaction with CRE-containing DNA. We report here crystal structures of a complex containing the CRTC2 CREB binding domain, the CREB basic leucine zipper domain and a CRE-containing DNA. The structures revealed that CRTC and CREB form a 2:2 complex on CRE-containing DNA, and CRTC interacts with both CREB and DNA through highly conserved residues. Structure-guided functional studies revealed that both interactions are crucial for the complex assembly and CREB stabilization on DNA. Interestingly, we found that the CRTC-DNA interaction confers selectivity toward the intrinsic DNA shape, which may play a role in selective transcription activation of the CRE genes.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Sitios de Unión , Núcleo Celular/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Modelos Moleculares , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Elementos de Respuesta , Transcripción Genética , Activación TranscripcionalRESUMEN
Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.
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Candida glabrata/enzimología , Proteínas Fúngicas/química , Sistema de la Enzima Desramificadora del Glucógeno/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Biocatálisis , Candida glabrata/genética , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucógeno/química , Glucógeno/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo III/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo III/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oligosacáridos/química , Oligosacáridos/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
All eukaryotic mRNAs are capped at their 5' end. Capping of mRNAs takes place co-transcriptionally and involves three steps. The intermediates of the capping process, as well as the uncapped 5' tri-phosphate RNA, are resistant to decapping and degradation by known factors, leading to the assumption that the capping process always proceeds to completion. This view was recently drastically changed. A novel family of enzymes, including the yeast proteins Rai1, Dxo1/Ydr370C, and the mammalian protein DXO/Dom3Z, has been identified. These enzymes catalyze the conversion of the improperly capped mRNAs to 5' mono-phosphate RNA, allowing them to be degraded by 5'-3' exoribonucleases. Several of these enzymes also possess 5'-3' exoribonuclease activities themselves, and can single-handedly clear the improperly capped mRNAs. Studying of these enzymes has led to the realization that mRNA capping does not always proceed to completion, and the identification of an mRNA capping quality control mechanism in eukaryotes. In this paper, we briefly review recent advances in this area.