Ethanol tolerance in engineered strains of Clostridium thermocellum.

Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75-80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

Erratum: Exploring APOBEC3A and APOBEC3B substrate specificity and their role in HPV positive head and neck cancer.

[This corrects the article DOI: 10.1016/j.isci.2022.105077.].

Exploring ABOBEC3A and APOBEC3B substrate specificity and their role in HPV positive head and neck cancer.

APOBEC3 family members are cytidine deaminases catalyzing conversion of cytidine to uracil. Many studies have established a link between APOBEC3 expression and cancer development and progression, especially APOBEC3A (A3A) and APOBEC3B (A3B). Preclinical studies with human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) and clinical trial specimens revealed induction of A3B, but not A3A expression after demethylation. We examined the kinetic features of the cytidine deaminase activity for full length A3B and found that longer substrates and a purine at -2 position favored by A3B, whereas A3A prefers shorter substrates and an adenine or thymine at -2 position. The importance and biological significance of A3B catalytic activity rather than A3A and a preference for purine at the -2 position was also established in HPV+ HNSCCs. Our study explored factors influencing formation of A3A and A3B-related cancer mutations that are essential for understanding APOBEC3-related carcinogenesis and facilitating drug discovery.

Enzymatic Synthesis of Xylan Microparticles with Tunable Morphologies.

Xylans are a diverse family of hemicellulosic polysaccharides found in abundance within the cell walls of nearly all flowering plants. Unfortunately, naturally occurring xylans are highly heterogeneous, limiting studies of their synthesis and structure-function relationships. Here, we demonstrate that xylan synthase 1 from the charophyte alga Klebsormidium flaccidum is a powerful biocatalytic tool for the bottom-up synthesis of pure ß-1,4 xylan polymers that self-assemble into microparticles in vitro. Using uridine diphosphate-xylose (UDP-xylose) and defined saccharide primers as substrates, we demonstrate that the shape, composition, and properties of the self-assembling xylan microparticles could be readily controlled via the fine structure of the xylan oligosaccharide primer used to initiate polymer elongation. Furthermore, we highlight two approaches for bottom-up and surface functionalization of xylan microparticles with chemical probes and explore the susceptibility of xylan microparticles to enzymatic hydrolysis. Together, these results provide a useful platform for structural and functional studies of xylans to investigate cell wall biosynthesis and polymer-polymer interactions and suggest possible routes to new biobased materials with favorable properties for biomedical and renewable applications.

Maedi-visna virus Vif protein uses motifs distinct from HIV-1 Vif to bind zinc and the cofactor required for A3 degradation.

The mammalian apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of cytidine deaminases restrict viral infections by mutating viral DNA and impeding reverse transcription. To overcome this antiviral activity, most lentiviruses express a viral accessory protein called the virion infectivity factor (Vif), which recruits A3 proteins to cullin-RING E3 ubiquitin ligases such as cullin-5 (Cul5) for ubiquitylation and subsequent proteasomal degradation. Although Vif proteins from primate lentiviruses such as HIV-1 utilize the transcription factor core-binding factor subunit beta as a noncanonical cofactor to stabilize the complex, the maedi-visna virus (MVV) Vif hijacks cyclophilin A (CypA) instead. Because core-binding factor subunit beta and CypA are both highly conserved among mammals, the requirement for two different cellular cofactors suggests that these two A3-targeting Vif proteins have different biochemical and structural properties. To investigate this topic, we used a combination of in vitro biochemical assays and in vivo A3 degradation assays to study motifs required for the MVV Vif to bind zinc ion, Cul5, and the cofactor CypA. Our results demonstrate that although some common motifs between the HIV-1 Vif and MVV Vif are involved in recruiting Cul5, different determinants in the MVV Vif are required for cofactor binding and stabilization of the E3 ligase complex, such as the zinc-binding motif and N- and C-terminal regions of the protein. Results from this study advance our understanding of the mechanism of MVV Vif recruitment of cellular factors and the evolution of lentiviral Vif proteins.

Advances in integrative structural biology: Towards understanding protein complexes in their cellular context.

Microorganisms rely on protein interactions to transmit signals, react to stimuli, and grow. One of the best ways to understand these protein interactions is through structural characterization. However, in the past, structural knowledge was limited to stable, high-affinity complexes that could be crystallized. Recent developments in structural biology have revolutionized how protein interactions are characterized. The combination of multiple techniques, known as integrative structural biology, has provided insight into how large protein complexes interact in their native environment. In this mini-review, we describe the past, present, and potential future of integrative structural biology as a tool for characterizing protein interactions in their cellular context.

Structural basis of antagonism of human APOBEC3F by HIV-1 Vif.

HIV-1 virion infectivity factor (Vif) promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion. Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of human immunodeficiency virus type 1 (HIV-1) replication is a potential approach for antiviral therapeutics. However, the molecular mechanisms by which Vif recognizes A3 proteins remain elusive. Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and the cellular cofactor core-binding factor beta (CBFß) at 3.9-Å resolution. The structure shows that Vif and CBFß form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFß beyond Vif stabilization, and captures multiple independent A3F-Vif interfaces. Together with our biochemical and cellular studies, our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F.

APOBEC3A Loop 1 Is a Determinant for Single-Stranded DNA Binding and Deamination.

The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of proteins functions in the innate immune system. The A3 proteins are interferon inducible and hypermutate deoxycytidine to deoxyuridine in foreign single-stranded DNA (ssDNA). However, this deaminase activity cannot discriminate between foreign and host ssDNA at the biochemical level, which presents a significant danger when A3 proteins gain access to the nucleus. Interestingly, this A3 capability can be harnessed when coupled with novel CRISPR-Cas9 proteins to create a targeted base editor. Specifically, A3A has been used in vitro to revert mutations associated with disease states. Recent structural studies have shown the importance of loop regions of A3A and A3G in ssDNA recognition and positioning for deamination. In this work, we further examined loop 1 of A3A to determine how it affects substrate selection, as well as the efficiency of deamination, in the hopes of advancing the potential of A3A in base editing technology. We found that mutating residue H29 enhanced deamination activity without changing substrate specificity. Also interestingly, we found that increasing the length of loop 1 decreases substrate specificity. Overall, these results lead to a better understanding of substrate recognition and deamination by A3A and the A3 family of proteins.

Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies.

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis-for example, A3G deaminates the third dC in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.