Assuntos
Cromossomos Humanos Par 1 , Neoplasias Meníngeas , Meningioma , Recidiva Local de Neoplasia , Humanos , Meningioma/genética , Meningioma/patologia , Cromossomos Humanos Par 1/genética , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Deleção Cromossômica , AdultoRESUMO
BACKGROUND: AL amyloidosis (AL) results from the misfolding of immunoglobulin light chains (IG LCs). Aim of this study was to comprehensively analyse kappa LC sequences from AL patients in comparison with multiple myeloma (MM). OBJECTIVE: We analysed IGKV/IGKJ usage and associated organ tropism and IGKV1/D-33 in terms of mutational analysis and theoretical biochemical properties. MATERIAL AND METHODS: cDNA and bulk RNA sequencing of the LCs of AL and MM patients. RESULTS: We studied 41 AL and 83 MM patients showing that IGKV1 was most expressed among kappa AL and MM, with higher frequency in AL (80% vs. 53%, p = .002). IGKV3 was underrepresented in AL (10% vs. 30%, p = .014). IGKJ2 was more commonly used in AL than in MM (39% vs. 29%). Patients with IGKV1/D-33 were associated with heart involvement (75%, p = .024). IGKV1/D-33-segments of AL had a higher mutation count (AL = 12.0 vs. MM = 10.0). FR3 and CDR3 were most frequently mutated in both, with a median mutation count in FR3 being the highest (AL = 4.0; MM = 3.5) and one mutation hotspot (FR3 (83I)) for IGKV1/D-33/IGKJ2 was associated with cardiac involvement. CONCLUSION: This study confirmed that germline usage has an influence on AL amyloidosis risk and organ involvement.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina , Cadeias kappa de Imunoglobulina , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Masculino , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Feminino , Pessoa de Meia-Idade , Cadeias kappa de Imunoglobulina/genética , Idoso , MutaçãoRESUMO
Light chain amyloidosis (AL) is one of the most common forms of systemic amyloidosis and is caused by the deposition of insoluble fibrils derived from misfolded and aggregated immunoglobulin light chains (LC). To uncover the causes leading to this aggregation, we compared AL LC sequences with those of patients with the related disease multiple myeloma (MM), which do not aggregate in insoluble fibrils in vivo. IGLV2-14 is one of the most common AL-associated IGLV subfamilies. Here, we analysed IGLV2-14 LC sequences of 13 AL and eight MM patients in detail. We found that AL-associated LCs presented a lower median mutation count (7.0 vs. 11.5 in MM; P = 0.045), as well as an overall composition of less charged amino acids than MM LCs. However, we did not find a mutation that was present in ≥ 50% of the AL and not in the MM sequences. Furthermore, we did not find a significant difference in the isoelectric point (pI) in general, suggesting similar stability of the LCs in AL and MM. However, the subgroup of patients without a detectable heavy chain stood out. Surprisingly, they are characterized by an increase in mutation count (median 7.0 vs. 5.5) and pI (median 7.82 vs. 6.44, P = 0.043). In conclusion, our data suggest that the amount of mutations and the introduction of charges play a crucial role in AL fibril formation, as well as the absence or presence of a potential heavy chain binding partner.
Assuntos
Amiloidose , Amiloidose de Cadeia Leve de Imunoglobulina , Mieloma Múltiplo , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Mieloma Múltiplo/genética , Amiloidose/genética , Amiloidose/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Mutação , Amiloide/químicaRESUMO
BACKGROUND: Systemic AL amyloidosis arises from the misfolding of patient-specific immunoglobulin light chains (LCs). Potential drivers of LC amyloid formation are mutational changes and post-translational modifications (PTMs). However, little information is available on the exact primary structure of the AL proteins and their precursor LCs. OBJECTIVE: We analyse the exact primary structure of AL proteins extracted from 10 λ AL amyloidosis patients and their corresponding precursor LCs. MATERIALS AND METHODS: By cDNA sequencing of the precursor LC genes in combination with mass spectrometry of the AL proteins, the exact primary structure and PTMs were determined. This information was used to analyse their biochemical properties. RESULTS: All AL proteins comprise the VL and a small part of the CL with a common C-terminal truncation region. While all AL proteins retain the conserved native disulphide bond of the VL, we found no evidence for presence of other common PTMs. The analysis of the biochemical properties revealed that the isoelectric point of the VL is significantly increased due to introduced mutations. CONCLUSION: Our data imply that mutational changes influence the surface charge properties of the VL and that common proteolytic processes are involved in the generation of the cleavage sites of AL proteins.
Assuntos
Amiloidose , Amiloidose de Cadeia Leve de Imunoglobulina , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose/genética , Amiloidose/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Amiloide/genética , Amiloide/metabolismo , Espectrometria de Massas , Gordura Abdominal/metabolismoRESUMO
Light chain amyloidosis is one of the most common forms of systemic amyloidosis. The disease is caused by the misfolding and aggregation of immunoglobulin light chains to insoluble fibrils. These fibrils can deposit in different tissues and organs such as heart and kidney and cause organ impairments that define the clinical presentation. In this study, we present an overview of IGLV-IGLJ and IGLC germline utilization in 85 patients classified in three clinically important subgroups with dominant cardiac, renal as well as cardiac and renal involvement. We found that IGLV3 was the most frequently detected IGLV-family in patients with dominant cardiac involvement, whereas in renal patients IGLV1 were most frequently identified. For patients with dominant heart and kidney involvement IGLV6 was the most frequently detected IGLV-family. In more detailed analysis IGLV3-21 was observed as the most dominant IGLV-subfamily for patients with dominant heart involvement and IGLV1-44 as the most frequent IGLV-subfamily in the group of patients with dominant kidney involvement. For patients with dominant heart and kidney involvement IGLV6-57 was the most frequently detected IGLV-subfamily. Additionally, we were able to show an exclusive linkage between IGLJ1 and IGLC1 as well as between IGLJ2 and IGLC2 in the fully assembled IGL mRNA.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina , Cadeias lambda de Imunoglobulina , Rim/metabolismo , Miocárdio/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Cadeias lambda de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/metabolismo , Masculino , Pessoa de Meia-Idade , Especificidade de ÓrgãosRESUMO
Systemic AL amyloidosis is a rare disease that is caused by the misfolding of immunoglobulin light chains (LCs). Potential drivers of amyloid formation in this disease are post-translational modifications (PTMs) and the mutational changes that are inserted into the LCs by somatic hypermutation. Here we present the cryo electron microscopy (cryo-EM) structure of an ex vivo λ1-AL amyloid fibril whose deposits disrupt the ordered cardiomyocyte structure in the heart. The fibril protein contains six mutational changes compared to the germ line and three PTMs (disulfide bond, N-glycosylation and pyroglutamylation). Our data imply that the disulfide bond, glycosylation and mutational changes contribute to determining the fibril protein fold and help to generate a fibril morphology that is able to withstand proteolytic degradation inside the body.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Microscopia Crioeletrônica , Glicosilação , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Mutação , Conformação Proteica , Dobramento de ProteínaRESUMO
Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.
Assuntos
Poliaminas/metabolismo , Xenorhabdus/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Vias Biossintéticas , Família Multigênica , Poliaminas/química , Xenorhabdus/química , Xenorhabdus/genéticaRESUMO
The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera Xenorhabdus or Photorhabdus. In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected Xenorhabdus and Photorhabdus mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine (fcl) biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS2 experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class.
RESUMO
Acetogenic bacteria are a group of strictly anaerobic bacteria that may have been first life forms on Earth since they employ an ancient pathway for CO2 fixation into acetyl-CoA that is coupled to the synthesis of ATP, the Wood-Ljungdahl pathway. Electrons for CO2 reduction are derived from oxidation of H2 or CO and thus, these bacteria can grow lithotrophically on gases present on early Earth. Among the organic molecules present on early Earth is acetaldehyde, a highly volatile C2 compound. Here, we demonstrate that the acetogenic model bacterium Acetobacterium woodii grows on acetaldehyde. Acetaldehyde is dismutated to ethanol and acetyl-CoA, most likely by the bifunctional alcohol dehydrogenase AdhE. Acetyl-CoA is converted to acetate by two subsequent enzymes, phosphotransacetylase and acetate kinase, accompanied by the synthesis of ATP by substrate-level phosphorylation. Apparently, growth on acetaldehyde does not employ the Wood-Ljungdahl pathway. Our finding opens the possibility of a simple and ancient metabolic pathway with only three enzymes that allows for biomass (acetyl-CoA) and ATP formation on early Earth.