Protein Interaction Map of APOBEC3 Enzyme Family Reveals Deamination-Independent Role in Cellular Function.

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.

Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure.

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.

Old Friend or New Ally: A Comparison of Follicular Unit Transplantation and Follicular Unit Excision Methods in Hair Transplantation.

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.

Follicular Unit Excision Punches and Devices.

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.

Enzyme cycling contributes to efficient induction of genome mutagenesis by the cytidine deaminase APOBEC3B.

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.

Mechanism of Enhanced HIV Restriction by Virion Coencapsidated Cytidine Deaminases APOBEC3F and APOBEC3G.

The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4+ T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart. IMPORTANCE: The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 Vif protein. After APOBEC3 enzymes are encapsidated into virions, they deaminate cytosines in minus-strand DNA, which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found, demonstrating that APOBEC3s still have discernible activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate that an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we still may not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif.

Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability.

APOBEC3H is a deoxycytidine deaminase that can restrict the replication of HIV-1 in the absence of the viral protein Vif that induces APOBEC3H degradation in cells. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Of the three stable APOBEC3H haplotypes (II, V, and VII), haplotypes II and V occur most frequently in the population. Despite APOBEC3H being a bona fide restriction factor, there has been no comparative biochemical characterization of APOBEC3H haplotypes. We characterized the ssDNA scanning mechanisms that haplotypes II and V use to search their ssDNA substrate for cytosine-containing deamination motifs. APOBEC3H haplotype II was able to processively deaminate multiple cytosines in a single enzyme-substrate encounter by using sliding, jumping, and intersegmental transfer movements. In contrast, APOBEC3H haplotype V exhibited diminished sliding and intersegmental transfer abilities but was able to jump along ssDNA. Due to an Asp or Glu at amino acid 178 differentiating these APOBEC3H haplotypes, the data indicated that this amino acid on helix 6 contributes to processivity. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assays, suggesting a redundancy in the contributions of jumping and intersegmental transfer to mutagenic efficiency. Optimal processivity on ssDNA also required dimerization of APOBEC3H through the ß2 strands. The findings support a model in which jumping can compensate for deficiencies in intersegmental transfer and suggest that APOBEC3H haplotypes II and V induce HIV-1 mutagenesis efficiently but by different mechanisms.

Different mutagenic potential of HIV-1 restriction factors APOBEC3G and APOBEC3F is determined by distinct single-stranded DNA scanning mechanisms.

The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (-)HIV-1 DNA. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an ¹9°NPM¹9² motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F ¹9¹Pro to ¹9¹Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F ¹9°NGM¹9² could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif (APOBEC3F, 5'TTC; APOBEC3G 5'CCC) influences the mutagenic and gene inactivation potential of an APOBEC3 enzyme.

HIV-1 viral infectivity factor (Vif) alters processive single-stranded DNA scanning of the retroviral restriction factor APOBEC3G.

APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (-)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of VifIIIB- and VifHXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, VifHXB2 inhibited the jumping and VifIIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.

Protein interaction map of APOBEC3 enzyme family reveals deamination-independent role in cellular function.

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.

Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A.

APOBEC3A belongs to a family of single-stranded DNA (ssDNA) DNA cytosine deaminases that are known for restriction of HIV through deamination-induced mutational inactivation, e.g. APOBEC3G, or initiation of somatic hypermutation and class switch recombination (activation-induced cytidine deaminase). APOBEC3A, which is localized to both the cytoplasm and nucleus, not only restricts HIV but can also initiate catabolism of cellular DNA. Despite being ascribed these roles, there is a paucity of data available on the biochemical mechanism by which APOBEC3A deaminates ssDNA. Here we assessed APOBEC3A deamination activity on ssDNA and in dynamic systems modeling HIV replication (cytoplasmic event) and DNA transcription (nuclear event). We find that APOBEC3A, unlike the highly processive APOBEC3G, exhibits low or no processivity when deaminating synthetic ssDNA substrates with two cytosines located 5-63 nucleotides apart, likely because of an apparent K(d) in the micromolar range (9.1 µm). APOBEC3A was able to deaminate nascently synthesized (-)DNA in an in vitro model HIV replication assay but induced fewer mutations overall in comparison to APOBEC3G. However, the data indicate that the target deamination motif (5'-TC for APOBEC3A and 5'-CC for APOBEC3G) and not the number of mutations best predicted the ability to mutationally inactivate HIV. We further assessed APOBEC3A for the ability to deaminate dsDNA undergoing transcription, which could allow for collateral deaminations to occur in genomic DNA similar to the action of activation-induced cytidine deaminase. That APOBEC3A was able to deaminate dsDNA undergoing transcription suggests a genomic cost of a deamination-based retroviral restriction system.

Developing Data Governance Agreements with Indigenous Communities in Canada: Toward Equitable Tuberculosis Programming, Research, and Reconciliation.

Indigenous rights to self-determination and data sovereignty support Indigenous-led data governance, which, when adequately resourced, can act as a catalyst for Indigenous-led strategic planning and decision-making in public health research and programming. Respecting Indigenous data sovereignty and governance requires time, resources, education, and planning. Here we share our experiences and lessons learned when developing and implementing data governance agreements with select First Nations and Métis partnering communities in Canada in the context of tuberculosis prevention and care. We define the process undertaken to create a decision space, supported by data governance agreements, where researchers, program (government) stakeholders, and Indigenous community partners are equally and equitably informed to co-develop public health interventions. The decision space has implications for tackling all manner of public health concerns and can inform policy for nation-to-nation public health relationships to advance public health goals.

Understanding the structural basis of HIV-1 restriction by the full length double-domain APOBEC3G.

APOBEC3G, a member of the double-domain cytidine deaminase (CD) APOBEC, binds RNA to package into virions and restrict HIV-1 through deamination-dependent or deamination-independent inhibition. Mainly due to lack of a full-length double-domain APOBEC structure, it is unknown how CD1/CD2 domains connect and how dimerization/multimerization is linked to RNA binding and virion packaging for HIV-1 restriction. We report rhesus macaque A3G structures that show different inter-domain packing through a short linker and refolding of CD2. The A3G dimer structure has a hydrophobic dimer-interface matching with that of the previously reported CD1 structure. A3G dimerization generates a surface with intensified positive electrostatic potentials (PEP) for RNA binding and dimer stabilization. Unexpectedly, mutating the PEP surface and the hydrophobic interface of A3G does not abolish virion packaging and HIV-1 restriction. The data support a model in which only one RNA-binding mode is critical for virion packaging and restriction of HIV-1 by A3G.

Advances in Hair Transplantation Technology: Multiphasic Device for Follicular Unit Excision.

Follicular unit excision (FUE) requires the careful excision of hair-bearing follicular units. The follicle can only be detached from the skin once the epidermis, dermis, and subcutaneous tissues are cut, and each layer requires a different amount of force. Traditional FUE devices can be set only to a single speed during a single excision. The Trivellini Device is a first-in-class programmable multiphasic device that is able to accommodate the cutting of various layers of tissue in a single program, while ensuring minimal transection of hair grafts. We detail the advances in the field of motorized FUE embodied by the multiphasic devices.

Polymorphisms of the cytidine deaminase APOBEC3F have different HIV-1 restriction efficiencies.

The APOBEC3 enzyme family are host restriction factors that induce mutagenesis of HIV-1 proviral genomes through the deamination of cytosine to form uracil in nascent single-stranded (-)DNA. HIV-1 suppresses APOBEC3 activity through the HIV-1 protein Vif that induces APOBEC3 degradation. Here we compared two common polymorphisms of APOBEC3F. We found that although both polymorphisms have HIV-1 restriction activity, APOBEC3F 108 A/231V can restrict HIV-1 ΔVif up to 4-fold more than APOBEC3F 108 S/231I and is partially protected from Vif-mediated degradation. This resulted from higher levels of steady state expression of APOBEC3F 108 A/231 V. Individuals are commonly heterozygous for the APOBEC3F polymorphisms and these polymorphisms formed in cells, independent of RNA, hetero-oligomers between each other and with APOBEC3G. Hetero-oligomerization with APOBEC3F 108 A/231V resulted in partial stabilization of APOBEC3F 108 S/231I and APOBEC3G in the presence of Vif. These data demonstrate functional outcomes of APOBEC3 polymorphisms and hetero-oligomerization that affect HIV-1 restriction.

Role of co-expressed APOBEC3F and APOBEC3G in inducing HIV-1 drug resistance.

The APOBEC3 enzymes can induce mutagenesis of HIV-1 proviral DNA through the deamination of cytosine. HIV-1 overcomes this restriction through the viral protein Vif that induces APOBEC3 proteasomal degradation. Within this dynamic host-pathogen relationship, the APOBEC3 enzymes have been found to be beneficial, neutral, or detrimental to HIV-1 biology. Here, we assessed the ability of co-expressed APOBEC3F and APOBEC3G to induce HIV-1 resistance to antiviral drugs. We found that co-expression of APOBEC3F and APOBEC3G enabled partial resistance of APOBEC3F to Vif-mediated degradation with a corresponding increase in APOBEC3F-induced deaminations in the presence of Vif, in addition to APOBEC3G-induced deaminations. We recovered HIV-1 drug resistant variants resulting from APOBEC3-induced mutagenesis, but these variants were less able to replicate than drug resistant viruses derived from RT-induced mutations alone. The data support a model in which APOBEC3 enzymes cooperate to restrict HIV-1, promoting viral inactivation over evolution to drug resistance.

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates.

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.

The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis.

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.