RESUMO
Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.
Assuntos
Citidina Desaminase , Mapas de Interação de Proteínas , Humanos , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Desaminação , Desaminases APOBEC/metabolismo , Aminoidrolases/metabolismo , Aminoidrolases/genética , Células HEK293 , Citosina Desaminase/metabolismo , Desaminase APOBEC-3G/metabolismo , Desaminase APOBEC-3G/genética , Spliceossomos/metabolismo , Ligação Proteica , Espectrometria de Massas , RNA/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Antígenos de Histocompatibilidade Menor/genéticaRESUMO
Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence is not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and map a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology.
RESUMO
BACKGROUND: Follicular unit excision (FUE) is used to harvest follicular units for hair transplantation using trephine punches. The characteristics of FUE punches can impact the success of this technique; thus, many innovative punch designs and devices have been developed. With many options available, it can be difficult for the hair restoration surgeon to know which punch best suits the needs of their patients. OBJECTIVE: To provide a comprehensive review of punch shapes and devices available. METHODS: Search of PubMed, reference mining of relevant publications, and hand searching trade publications. RESULTS: We examined FUE punches and devices and consolidated descriptive information for each to create textual and visual guides. No single punch shape or device may suit all cases; thus, it is important to know the best uses and limitations of each. CONCLUSION: The surgeon should have a comprehensive knowledge base of available punch shapes and devices and understand the advantages and disadvantages of each. It is also beneficial to have an in-depth knowledge of skin properties and follicular unit structure. Ultimately, understanding the dynamics behind punch excision will enhance the FUE technique.
Assuntos
Alopecia/cirurgia , Folículo Piloso/transplante , Coleta de Tecidos e Órgãos/instrumentação , Desenho de Equipamento , Humanos , Transplante Autólogo/instrumentaçãoRESUMO
BACKGROUND: Follicular unit excision (FUE) and follicular unit transplantation using strip surgery (FUT) are the dominant graft harvest methods in hair transplantation. The increase in the demand for FUE has reignited the debate of the relative superiority of the 2 methods. OBJECTIVE: To present a critical comparison of FUE and FUT graft harvesting techniques. MATERIALS AND METHODS: Search of PubMed, trade publications, and printed references. RESULTS: Follicular unit excision and FUT methods provide high-quality grafts, but differ in their scarring patterns of the donor region. Follicular unit transplantation results in a linear scar, whereas FUE produces punctate scars that are typically easily concealed. Distinct subgroups of hair transplant patients are eligible for FUE, FUT, or both procedures. CONLCUSION: Both FUE and FUT are equally effective in generating high-quality grafts. This detailed evaluation of the FUT and FUE procedures will assist hair restoration surgeons make informed decisions about the best approach for their patients.
Assuntos
Folículo Piloso/transplante , Coleta de Tecidos e Órgãos/métodos , Cicatriz/etiologia , Sobrevivência de Enxerto , Humanos , Preferência do Paciente , Reoperação , Deiscência da Ferida Operatória/etiologia , Coleta de Tecidos e Órgãos/efeitos adversos , Transplante Autólogo/efeitos adversos , Transplante Autólogo/métodosRESUMO
The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5'TC3' or 5'CT3' dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.
Assuntos
Citidina Desaminase/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Desaminação/genética , Desaminases APOBEC , Sítios de Ligação/genética , Linhagem Celular , Citidina Desaminase/química , Citosina Desaminase/química , Citosina Desaminase/genética , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , HIV-1/genética , HIV-1/patogenicidade , Vírus da Hepatite B/genética , Humanos , Mutagênese/genética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , Retroelementos/genéticaRESUMO
The Apolipoprotein B mRNA editing complex (APOBEC) family of enzymes contains single-stranded polynucleotide cytidine deaminases. These enzymes catalyze the deamination of cytidine in RNA or single-stranded DNA, which forms uracil. From this 11 member enzyme family in humans, the deamination of single-stranded DNA by the seven APOBEC3 family members is considered here. The APOBEC3 family has many roles, such as restricting endogenous and exogenous retrovirus replication and retrotransposon insertion events and reducing DNA-induced inflammation. Similar to other APOBEC family members, the APOBEC3 enzymes are a double-edged sword that can catalyze deamination of cytosine in genomic DNA, which results in potential genomic instability due to the many mutagenic fates of uracil in DNA. Here, we discuss how these enzymes find their single-stranded DNA substrate in different biological contexts such as during human immunodeficiency virus (HIV) proviral DNA synthesis, retrotransposition of the LINE-1 element, and the "off-target" genomic DNA substrate. The enzymes must be able to efficiently deaminate transiently available single-stranded DNA during reverse transcription, replication, or transcription. Specific biochemical characteristics promote deamination in each situation to increase enzyme efficiency through processivity, rapid enzyme cycling between substrates, or oligomerization state. The use of biochemical data to clarify biological functions and alignment with cellular data is discussed. Models to bridge knowledge from biochemical, structural, and single molecule experiments are presented.
Assuntos
Citosina Desaminase/metabolismo , DNA de Cadeia Simples/metabolismo , Desaminases APOBEC , Citidina Desaminase , Replicação do DNA , HIV/fisiologia , Humanos , Provírus/fisiologia , Recombinação Genética , RetroelementosRESUMO
The single-stranded DNA cytidine deaminases APOBEC3B, APOBEC3H haplotype I, and APOBEC3A can contribute to cancer through deamination of cytosine to form promutagenic uracil in genomic DNA. The enzymes must access single-stranded DNA during the dynamic processes of DNA replication or transcription, but the enzymatic mechanisms enabling this activity are not known. To study this, we developed a method to purify full length APOBEC3B and characterized it in comparison to APOBEC3A and APOBEC3H on substrates relevant to cancer mutagenesis. We found that the ability of an APOBEC3 to cycle between DNA substrates determined whether it was able to efficiently deaminate single-stranded DNA produced by replication and single-stranded DNA bound by replication protein A (RPA). APOBEC3 deaminase activity during transcription had a size limitation that inhibited APOBEC3B tetramers, but not APOBEC3A monomers or APOBEC3H dimers. Altogether, the data support a model in which the availability of single-stranded DNA is necessary, but alone not sufficient for APOBEC3-induced mutagenesis in cells because there is also a dependence on the inherent biochemical properties of the enzymes. The biochemical properties identified in this study can be used to measure the mutagenic potential of other APOBEC enzymes in the genome.
Assuntos
Citidina Desaminase/genética , DNA de Cadeia Simples/genética , Genoma Humano/genética , Antígenos de Histocompatibilidade Menor/genética , Mutagênese , Animais , Biocatálise , Citidina Desaminase/química , Citidina Desaminase/metabolismo , Citosina/química , Citosina/metabolismo , DNA de Cadeia Simples/metabolismo , Desaminação , Humanos , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Ligação Proteica , Multimerização Proteica , Proteína de Replicação A/metabolismo , Células Sf9 , Spodoptera , Especificidade por Substrato , Uracila/química , Uracila/metabolismoRESUMO
Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of 'APOBEC signature' mutations in cancer.