RESUMEN
Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.
Asunto(s)
Empaquetamiento del ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/química , Empaquetamiento del Genoma Viral , Proteínas Virales/química , Fagos de Bacillus/química , Fagos de Bacillus/genética , Microscopía por Crioelectrón , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Esterasas/química , Evolución Molecular , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios Proteicos , ARN Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Pulmonary masses occasionally occur after lung transplantation and vary in etiology, which includes malignant and benign conditions, such as infection. Here, we report a case of a patient presenting with a lung mass 3 years after lung transplant. To our knowledge, this is the first described case of pulmonary malakoplakia due to Rhodococcus equi infection in an allograft post-lung transplantation. This case outlines the challenges of differentiating benign from malignant masses after transplantation.
Asunto(s)
Infecciones por Actinomycetales/complicaciones , Enfermedades Pulmonares/diagnóstico , Neoplasias Pulmonares/diagnóstico , Trasplante de Pulmón/efectos adversos , Malacoplasia/diagnóstico , Complicaciones Posoperatorias , Rhodococcus equi/aislamiento & purificación , Infecciones por Actinomycetales/epidemiología , Infecciones por Actinomycetales/microbiología , Anciano , Diagnóstico Diferencial , Humanos , Enfermedades Pulmonares/etiología , Neoplasias Pulmonares/etiología , Malacoplasia/etiología , Masculino , PronósticoRESUMEN
The immune inhibitory enzyme indoleamine 2,3-dioxygenase (IDO) has been associated with immune evasion in numerous malignancies and may mark these cancers as susceptible to anti-IDO therapies. We herein address IDO expression in breast cancers, examine the relationship between IDO and PD-L1, and investigate IDO fidelity across breast cancer primaries and metastases. IDO and PD-L1 expression was assessed in tissue microarrays containing 242 invasive primary breast cancers, 20 nodal metastases, and 19 distant metastases. IDO and PD-L1 were scored by extent in the tumor cells and immune infiltrate. Tumor IDO staining was seen in 14% of primaries including 38% of triple-negative cancers. IDO immune cell staining was seen in 14% of primaries and 29% of triple-negative cancers. Tumoral IDO and PD-L1 co-expression was seen in 8% of primaries, including 70% of tumoral PD-L1-positive cases. Immune IDO and PD-L1 co-expression was identified in 14% of primaries, including 48% of immune PD-L1-positive cases. Tumoral and immune cell IDO was conserved in 94% of matched primary/metastasis. In summary, IDO expression is common among high-grade, triple-negative breast cancers and is frequently associated with PD-L1 co-expression, suggesting that IDO might be a mechanism of anti-PD-1/PD-L1 immunotherapy resistance and that dual therapy may be of utility. Tumoral and immune cell IDO expression shows fidelity between primary and metastatic sites in treatment-naïve cancers, arguing against IDO as an independent driver for metastatic spread. Clinical trials are needed to assess the efficacy of IDO inhibition relative to IDO expression, as well as its possible role in combination with anti-PD-1/PD-L1 immunotherapy.
Asunto(s)
Antígeno B7-H1/biosíntesis , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Antígeno B7-H1/análisis , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/análisisRESUMEN
Suppression of the immune system is intimately linked to the development and progression of malignancy, and immune modulating treatment options have shown promise in a variety of tumor types, including some triple-negative breast cancers (TNBC). The most dramatic therapeutic success has been seen with immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and its ligand, PD-L1. Difficulty remains, however, in appropriate patient selection for treatment, as many PD-L1-positive cancers fail to show durable responses to PD-1/PD-L1 inhibition. Checkpoint inhibitor targeting of the adaptive immune response relies on the presence of major histocompatibility complex (MHC) class I molecules on the tumor cell surface for tumor antigen presentation. MHC class I loss has been previously described in breast cancer and represents a putative mechanism of immunotherapeutic resistance in this tumor type. One hundred seventeen invasive primary breast carcinomas with a range of histologic subtypes were evaluated on tissue microarrays containing formalin-fixed paraffin-embedded tissue. Loss of MHC class I expression was common among breast cancers, with greater than half of cases demonstrating either subclonal or diffuse loss. Fifty-nine percent of TNBC demonstrated loss of MHC class I, including 46% of those meeting the Food and Drug Administration-approved threshold of 1% for tumor-associated immune cell PD-L1 expression. MHC class I loss was particularly common in the apocrine subtype of TNBC (78%). MHC class I's employment as a predictive biomarker should be considered, as its loss may represent a barrier to successful enhancement of the antitumor adaptive immune response by PD-1/PD-L1 inhibition.
Asunto(s)
Antígeno B7-H1/análisis , Biomarcadores de Tumor/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Receptor de Muerte Celular Programada 1/análisis , Neoplasias de la Mama Triple Negativas/inmunología , Inmunidad Adaptativa , Adulto , Anciano , Anciano de 80 o más Años , Antígeno B7-H1/antagonistas & inhibidores , Biomarcadores de Tumor/antagonistas & inhibidores , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunohistoquímica , Persona de Mediana Edad , Selección de Paciente , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Análisis de Matrices Tisulares , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Microambiente TumoralRESUMEN
Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.
Asunto(s)
Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Fenómenos Químicos , Empaquetamiento del ADN , ADN Viral , Lactococcus lactis/virología , Adenosina Trifosfatasas , Bacteriófagos/ultraestructura , Clonación Molecular , Expresión Génica , Modelos Moleculares , Proteínas Recombinantes , Análisis Espectral , Relación Estructura-Actividad , Estruvita , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/ultraestructura , Ensamble de VirusRESUMEN
OBJECTIVES: Although the role of human papillomavirus (HPV) in the development of some carcinomas (eg, anogenital and oropharyngeal squamous cell carcinomas) is nondebatable, there is still significant controversy regarding the relationship of HPV and esophageal squamous cell carcinomas (SCCs). METHODS: All cases were sampled at or near the gastroesophageal junctions in patients with reflux and/or known Barrett esophagus and appear to have been initially sampled "incidentally." Patients were all men, aged 56 to 80 years. None had a known history of other HPV-related disease. RESULTS: We present four cases of high-grade squamous intraepithelial lesion of the gastroesophageal junction secondary to high-risk HPV that have identical histologic features to similar lesions of the anogenital tract. CONCLUSIONS: Whether such lesions are at risk for developing into invasive SCC remains unclear.
Asunto(s)
Enfermedades del Esófago/virología , Unión Esofagogástrica/virología , Infecciones por Papillomavirus/complicaciones , Lesiones Intraepiteliales Escamosas/virología , Anciano de 80 o más Años , Enfermedades del Esófago/patología , Unión Esofagogástrica/patología , Humanos , Masculino , Persona de Mediana Edad , Papillomaviridae , Lesiones Intraepiteliales Escamosas/patologíaRESUMEN
Mismatch repair (MMR) deficiency in solid tumors has recently been linked to susceptibility to immunotherapies targeting the programmed cell death-1 (PD-1)/programmed cell death-1 ligand (PD-L1) axis. Loss of MMR proteins has been shown to correlate with tumoral PD-L1 expression in colorectal and endometrial carcinomas, but the association between expression of MMR proteins and PD-L1 has not previously been studied in breast carcinoma, where MMR deficiency is less common. We assessed the relationship between PD-L1 and MMR protein expression by immunohistochemistry in 245 primary and 40 metastatic breast carcinomas. Tumoral staining for PD-L1 was positive in 12% of all cases, including 32% of triple-negative cancers. MMR deficiency was observed in 0.04% of breast cancers; the single MMR-deficient case was a high-grade, triple-negative ductal carcinoma which showed dual loss of MLH1 and PMS2 proteins and expressed PD-L1. Two ER carcinomas initially were scored with MMR protein loss in tissue microarray format but were subsequently shown to be MMR-intact on whole sections. Analysis of MMR gene mutation in The Cancer Genome Atlas corroborates low frequency of MMR deficiency for invasive breast cancer. MMR protein expression is therefore unlikely to show utility as a screen for immunotherapeutic vulnerability in this tumor type, and may provoke unwarranted genetic testing in patients unlikely to have a heritable cancer syndrome. PD-L1 may be a more clinically relevant biomarker for anti-PD-1/PD-L1 therapies in this setting.
Asunto(s)
Antígeno B7-H1/análisis , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Carcinoma/química , Reparación de la Incompatibilidad de ADN , Enzimas Reparadoras del ADN/análisis , Anciano , Biomarcadores de Tumor/genética , Biopsia , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Carcinoma/genética , Carcinoma/secundario , Carcinoma/cirugía , Enzimas Reparadoras del ADN/genética , Bases de Datos Genéticas , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/análisis , Homólogo 1 de la Proteína MutL/análisis , Mutación , Clasificación del Tumor , Valor Predictivo de las PruebasRESUMEN
Tumor expression of programmed cell death ligand 1 (PD-L1) is associated with immune evasion in a variety of malignancies, including a subset of triple-negative breast carcinomas, and may mark cancers as susceptible to PD-1/PD-L1 inhibitor therapies. We herein characterize PD-L1 expression in breast cancers across the full range of histomorphologies and investigate its intratumoral heterogeneity and fidelity across primaries and metastases. A total of 245 primary and 40 metastatic (20 nodal, 20 distant) breast carcinomas were evaluated with PD-L1 immunohistochemistry on tissue microarray. Tumor PD-L1 staining was seen in 12% of all primaries including 32% of triple-negative cancers. Staining was common in ductal cancers with medullary (54%), apocrine (27%), and metaplastic features (40%). However, diffuse (>50%) staining was rare (2% of all cancers and 5% of triple negatives). Immune staining was seen in 29% of all primaries and 61% of triple negatives. Tumor expression of PD-L1 was conserved in 94% of matched primary/metastasis pairs, while immune staining showed fidelity in 71%; the remaining cases acquired PD-L1 immune cell expression in the metastasis. Only half of cases with positive tumor staining showed concordance across all analyzed cores. These data demonstrate that PD-L1 expression is prevalent among high-grade, hormone receptor-negative breast cancers with a range of histomorphologies and shows fidelity between primary and metastatic sites in treatment-naive cancers, although acquisition of immune PD-L1 staining in metastases is not uncommon. There is considerable intratumoral heterogeneity in PD-L1 expression, undermining the suitability of core biopsy in the determination of PD-L1 status. Clinical trials are needed to determine PD-L1 staining thresholds required for therapeutic response, as diffuse staining is rare.