New approaches to predict the effect of co-occurring variants on protein characteristics.

Predicting the effect of a mutated gene before the onset of symptoms of genetic diseases would greatly facilitate diagnosis and potentiate early intervention. There have been myriad attempts to predict the effects of single-nucleotide variants. However, the applicability of these efforts does not scale to co-occurring variants. Furthermore, an increasing number of protein therapeutics contain co-occurring nucleotide variations, adding uncertainty during development to the safety and efficiency of these drugs. Co-occurring nucleotide variants may often have synergistic, additive, or antagonistic effects on protein attributes, further complicating the task of outcome prediction. We tested four models based on the cooperative and antagonistic effects of co-occurring variants to predict pathogenicity and effectiveness of protein therapeutics. A total of 30 attributes, including amino acid and nucleotide features, as well as existing single-variant effect prediction tools, were considered on the basis of previous studies on single-nucleotide variants. Importantly, the effects of synonymous variants, often seen in protein therapeutics, were also included in our models. We used 12 datasets of people with monogenic diseases and controls with co-occurring genetic variants to evaluate the accuracy of our models, accomplishing a degree of accuracy comparable to that of prediction tools for single-nucleotide variants. More importantly, our framework is generalizable to new, well-curated datasets of monogenic diseases and new variant scoring tools. This approach successfully assists in addressing the challenging task of predicting the effect of co-occurring variants on pathogenicity and protein effectiveness and is applicable for a wide range of protein therapeutics and genetic diseases.

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis is a recognized complication of Coronavirus disease of 2019 (COVID-19) and is often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms, by which some of these variants may contribute to disease, are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.

De Novo Sphingolipid Biosynthesis Is Required for Adipocyte Survival and Metabolic Homeostasis.

Sphingolipids are a diverse class of essential cellular lipids that function as structural membrane components and as signaling molecules. Cells acquire sphingolipids by both de novo biosynthesis and recycling of exogenous sphingolipids. The individual importance of these pathways for the generation of essential sphingolipids in differentiated cells is not well understood. To investigate the requirement for de novo sphingolipid biosynthesis in adipocytes, a cell type with highly regulated lipid metabolism, we generated mice with an adipocyte-specific deletion of Sptlc1 Sptlc1 is an obligate subunit of serine palmitoyltransferase, the enzyme responsible for the first and rate-limiting step of de novo sphingolipid biosynthesis. These mice, which initially developed adipose tissue, exhibited a striking age-dependent loss of adipose tissue accompanied by evidence of adipocyte death, increased macrophage infiltration, and tissue fibrosis. Adipocyte differentiation was not affected by the Sptlc1 deletion. The mice also had elevated fasting blood glucose, fatty liver, and insulin resistance. Collectively, these data indicate that de novo sphingolipid biosynthesis is required for adipocyte cell viability and normal metabolic function and that reduced de novo sphingolipid biosynthesis within adipocytes is associated with adipocyte death, adipose tissue remodeling, and metabolic dysfunction.

A new and updated resource for codon usage tables.

BACKGROUND: Due to the degeneracy of the genetic code, most amino acids can be encoded by multiple synonymous codons. Synonymous codons naturally occur with different frequencies in different organisms. The choice of codons may affect protein expression, structure, and function. Recombinant gene technologies commonly take advantage of the former effect by implementing a technique termed codon optimization, in which codons are replaced with synonymous ones in order to increase protein expression. This technique relies on the accurate knowledge of codon usage frequencies. Accurately quantifying codon usage bias for different organisms is useful not only for codon optimization, but also for evolutionary and translation studies: phylogenetic relations of organisms, and host-pathogen co-evolution relationships, may be explored through their codon usage similarities. Furthermore, codon usage has been shown to affect protein structure and function through interfering with translation kinetics, and cotranslational protein folding. RESULTS: Despite the obvious need for accurate codon usage tables, currently available resources are either limited in scope, encompassing only organisms from specific domains of life, or greatly outdated. Taking advantage of the exponential growth of GenBank and the creation of NCBI's RefSeq database, we have developed a new database, the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs), to present and analyse codon usage tables for every organism with publicly available sequencing data. Compared to existing databases, this new database is more comprehensive, addresses concerns that limited the accuracy of earlier databases, and provides several new functionalities, such as the ability to view and compare codon usage between individual organisms and across taxonomical clades, through graphical representation or through commonly used indices. In addition, it is being routinely updated to keep up with the continuous flow of new data in GenBank and RefSeq. CONCLUSION: Given the impact of codon usage bias on recombinant gene technologies, this database will facilitate effective development and review of recombinant drug products and will be instrumental in a wide area of biological research. The database is available at hive.biochemistry.gwu.edu/review/codon .

Expression of the ORMDLS, modulators of serine palmitoyltransferase, is regulated by sphingolipids in mammalian cells.

The relationship between serine palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.

Autophagy regulates sphingolipid levels in the liver.

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.

Functional properties of the HIV-1 long terminal repeat containing single-nucleotide polymorphisms in Sp site III and CCAAT/enhancer binding protein site I.

BACKGROUND: HIV-1 gene expression is driven by the long terminal repeat (LTR), which contains many binding sites shown to interact with an array of host and viral factors. Selective pressures within the host as well as the low fidelity of reverse transcriptase lead to changes in the relative prevalence of genetic variants within the HIV-1 genome, including the LTR, resulting in viral quasispecies that can be differentially regulated and can potentially establish niches within specific cell types and tissues. METHODS: Utilizing flow cytometry and electromobility shift assays, specific single-nucleotide sequence polymorphisms (SNPs) were shown to alter both the phenotype of LTR-driven transcription and reactivation. Additional studies also demonstrated differential loading of transcription factors to probes derived from the double-variant LTR as compared to probes from the wild type. RESULTS: This study has identified specific SNPs within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3 T, C-to-T change at position 3, and 5 T, C-to-T change at position 5 of the binding site, respectively) that alter LTR-driven gene transcription and may alter the course of viral latency and reactivation. The HIV-1 LAI LTRs containing the SNPs of interest were coupled to a plasmid encoding green fluorescent protein (GFP), and polyclonal HIV-1 LTR-GFP stable cell lines utilizing bone marrow progenitor, T, and monocytic cell lines were constructed and utilized to explore the LTR phenotype associated with these genotypic changes. CONCLUSIONS: Although the 3 T and 5 T SNPs have been shown to be low-affinity binding sites, the fact that they can still result in effective HIV-1 LTR-driven gene expression, particularly within the TF-1 cell line, has suggested that the low binding site affinities associated with the 3 T C/EBP site I and 5 T Sp site III are potentially compensated for by the interaction of nuclear factor-κB with its corresponding binding sites under selected physiological and cellular conditions. Additionally, tumor necrosis factor-α and Tat can enhance basal transcription of each SNP-specific HIV-1 LTR; however, differential regulation of the LTR is both SNP- and cell type-specific.

Novel Immunotherapeutics: Perspectives on Checkpoints, Bispecifics, and Vaccines in Development.

Over the past decade, the advent of molecular techniques and deeper understanding of the tumor microenvironment (TME) have enabled the development of a multitude of immunotherapy targets and approaches. Despite the revolutionary advancement in immunotherapy, treatment resistance remains a challenge leading to decreased response rate in a significant proportion of patients. As such, there has recently been an evolving focus to enhance efficacy, durability, and toxicity profiles of immunotherapy. Although immune checkpoint inhibitors have revolutionized cancer treatment with many already-approved antibodies and several others in the pipeline, bispecific antibodies build on their success in an attempt to deliver an even more potent immune response against tumor cells. On the other hand, vaccines comprise the oldest and most versatile form of immunotherapy. Peptide and nucleic acid vaccines are relatively simple to manufacture compared with oncolytic virus-based vaccines, whereas the dendritic cell vaccines are the most complex, requiring autologous cell culture. Nevertheless, a crucial question in the development of cancer vaccines is the choice of antigen whereby shared and patient-private antigen approaches are currently being pursued. There is hope that cancer vaccines will join the repertoire of successful novel immunotherapeutics in the market. Better insights into the impact of immunotherapy on effector T cells and other immune cell populations in the TME shall be a major priority across the immune-oncology discipline and can help identify predictive biomarkers to evaluate response to treatment and identify patients who would most likely benefit from immunotherapy.

Structural, functional, and immunogenicity implications of F9 gene recoding.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.

Distinct signatures of codon and codon pair usage in 32 primary tumor types in the novel database CancerCoCoPUTs for cancer-specific codon usage.

BACKGROUND: Gene expression is highly variable across tissues of multi-cellular organisms, influencing the codon usage of the tissue-specific transcriptome. Cancer disrupts the gene expression pattern of healthy tissue resulting in altered codon usage preferences. The topic of codon usage changes as they relate to codon demand, and tRNA supply in cancer is of growing interest. METHODS: We analyzed transcriptome-weighted codon and codon pair usage based on The Cancer Genome Atlas (TCGA) RNA-seq data from 6427 solid tumor samples and 632 normal tissue samples. This dataset represents 32 cancer types affecting 11 distinct tissues. Our analysis focused on tissues that give rise to multiple solid tumor types and cancer types that are present in multiple tissues. RESULTS: We identified distinct patterns of synonymous codon usage changes for different cancer types affecting the same tissue. For example, a substantial increase in GGT-glycine was observed in invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), and mixed invasive ductal and lobular carcinoma (IDLC) of the breast. Change in synonymous codon preference favoring GGT correlated with change in synonymous codon preference against GGC in IDC and IDLC, but not in ILC. Furthermore, we examined the codon usage changes between paired healthy/tumor tissue from the same patient. Using clinical data from TCGA, we conducted a survival analysis of patients based on the degree of change between healthy and tumor-specific codon usage, revealing an association between larger changes and increased mortality. We have also created a database that contains cancer-specific codon and codon pair usage data for cancer types derived from TCGA, which represents a comprehensive tool for codon-usage-oriented cancer research. CONCLUSIONS: Based on data from TCGA, we have highlighted tumor type-specific signatures of codon and codon pair usage. Paired data revealed variable changes to codon usage patterns, which must be considered when designing personalized cancer treatments. The associated database, CancerCoCoPUTs, represents a comprehensive resource for codon and codon pair usage in cancer and is available at https://dnahive.fda.gov/review/cancercocoputs/ . These findings are important to understand the relationship between tRNA supply and codon demand in cancer states and could help guide the development of new cancer therapeutics.

HIV-1 infection of bone marrow hematopoietic progenitor cells and their role in trafficking and viral dissemination.

Patients with HIV-1 often present with a wide range of hematopoietic abnormalities, some of which may be due to the presence of opportunistic infections and to therapeutic drug treatments. However, many of these abnormalities are directly related to HIV-1 replication in the bone marrow (BM). Although the most primitive hematopoietic progenitor cells (HPCs) are resistant to HIV-1 infection, once these cells begin to differentiate and become committed HPCs they become increasingly susceptible to HIV-1 infection and permissive to viral gene expression and infectious virus production. Trafficking of BM-derived HIV-1-infected monocytes has been shown to be involved in the dissemination of HIV-1 into the central nervous system (CNS), and it is possible that HIV-1 replication in the BM and infection of BM HPCs may be involved in the early steps leading to the development of HIV-1-associated dementia (HAD) as an end result of this cellular trafficking process. In addition, the growth and development of HPCs in the BM of patients with HIV-1 has also been shown to be impaired due to the presence of HIV-1 proteins and changes in the cytokine milieu, potentially leading to an altered maturation process and to increased cell death within one or more BM cell lineages. Changes in the growth and differentiation process of HPCs may be involved in the generation of monocyte populations that are more susceptible and/or permissive to HIV-1, and have potentially altered trafficking profiles to several organs, including the CNS. A monocyte subpopulation with these features has been shown to expand during the course of HIV-1 disease, particularly in HAD patients, and is characterized by low CD14 expression and the presence of cell surface CD16.

Immunogenicity of Protein Therapeutics: A Lymph Node Perspective.

The continuous development of molecular biology and protein engineering technologies enables the expansion of the breadth and complexity of protein therapeutics for in vivo administration. However, the immunogenicity and associated in vivo development of antibodies against therapeutics are a major restriction factor for their usage. The B cell follicular and particularly germinal center areas in secondary lymphoid organs are the anatomical sites where the development of antibody responses against pathogens and immunogens takes place. A growing body of data has revealed the importance of the orchestrated function of highly differentiated adaptive immunity cells, including follicular helper CD4 T cells and germinal center B cells, for the optimal generation of these antibody responses. Understanding the cellular and molecular mechanisms mediating the antibody responses against therapeutics could lead to novel strategies to reduce their immunogenicity and increase their efficacy.

Potential impact on coagulopathy of gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis has been one of the complications of the Coronavirus disease of 2019 (COVID-19), often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms by which some of these variants may contribute to disease are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches. AUTHOR SUMMARY: Increased blood clotting, especially in the lungs, is a common complication of COVID-19. Infectious diseases cause inflammation which in turn can contribute to increased blood clotting. However, the extent of clot formation that is seen in the lungs of COVID-19 patients suggests that there may be a more direct link. We identified three human proteins that are involved indirectly in the blood clotting cascade and have been shown to interact with proteins of SARS virus, which is closely related to the novel coronavirus. We examined computationally the interaction of these human proteins with the viral proteins. We looked for genetic variants of these proteins and examined how these variants are distributed across populations. We investigated whether variants of these genes could impact severity of COVID-19. Further investigation around these variants may provide clues for the pathogenesis of COVID-19 particularly in minority groups.

Coagulopathy and Thrombosis as a Result of Severe COVID-19 Infection: A Microvascular Focus.

Coronavirus disease of 2019 (COVID-19) is the clinical manifestation of the respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While primarily recognized as a respiratory disease, it is clear that COVID-19 is systemic illness impacting multiple organ systems. One defining clinical feature of COVID-19 has been the high incidence of thrombotic events. The underlying processes and risk factors for the occurrence of thrombotic events in COVID-19 remain inadequately understood. While severe bacterial, viral, or fungal infections are well recognized to activate the coagulation system, COVID-19-associated coagulopathy is likely to have unique mechanistic features. Inflammatory-driven processes are likely primary drivers of coagulopathy in COVID-19, but the exact mechanisms linking inflammation to dysregulated hemostasis and thrombosis are yet to be delineated. Cumulative findings of microvascular thrombosis has raised question if the endothelium and microvasculature should be a point of investigative focus. von Willebrand factor (VWF) and its protease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), play important role in the maintenance of microvascular hemostasis. In inflammatory conditions, imbalanced VWF-ADAMTS-13 characterized by elevated VWF levels and inhibited and/or reduced activity of ADAMTS-13 has been reported. Also, an imbalance between ADAMTS-13 activity and VWF antigen is associated with organ dysfunction and death in patients with systemic inflammation. A thorough understanding of VWF-ADAMTS-13 interactions during early and advanced phases of COVID-19 could help better define the pathophysiology, guide thromboprophylaxis and treatment, and improve clinical prognosis.

Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design.

As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses, for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.

Ribosome profiling of HEK293T cells overexpressing codon optimized coagulation factor IX.

Ribosome profiling provides the opportunity to evaluate translation kinetics at codon level resolution. Here, we describe ribosome profiling data, generated from two HEK293T cell lines. The ribosome profiling data are composed of Ribo-seq (mRNA sequencing data from ribosome protected fragments) and RNA-seq data (total RNA sequencing). The two HEK293T cell lines each express a version of the F9 gene, both of which are translated into identical proteins in terms of their amino acid sequences. However, these F9 genes vary drastically in their codon usage and predicted mRNA structure. We also provide the pipeline that we used to analyze the data. Further analyzing this dataset holds great potential as it can be used i) to unveil insights into the composition and regulation of the transcriptome, ii) for comparison with other ribosome profiling datasets, iii) to measure the rate of protein synthesis across the proteome and identify differences in elongation rates, iv) to discover previously unidentified translation of peptides, v) to explore the effects of codon usage or codon context in translational kinetics and vi) to investigate cotranslational folding. Importantly, a unique feature of this dataset, compared to other available ribosome profiling data, is the presence of the F9 gene in two very distinct coding sequences.

Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design.

As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.

TissueCoCoPUTs: Novel Human Tissue-Specific Codon and Codon-Pair Usage Tables Based on Differential Tissue Gene Expression.

Protein expression in multicellular organisms varies widely across tissues. Codon usage in the transcriptome of each tissue is derived from genomic codon usage and the relative expression level of each gene. We created a comprehensive computational resource that houses tissue-specific codon, codon-pair, and dinucleotide usage data for 51 Homo sapiens tissues (TissueCoCoPUTs: https://hive.biochemistry.gwu.edu/review/tissue_codon), using transcriptome data from the Broad Institute Genotype-Tissue Expression (GTEx) portal. Distances between tissue-specific codon and codon-pair frequencies were used to generate a dendrogram based on the unique patterns of codon and codon-pair usage in each tissue that are clearly distinct from the genomic distribution. This novel resource may be useful in unraveling the relationship between codon usage and tRNA abundance, which could be critical in determining translation kinetics and efficiency across tissues. Areas of investigation such as biotherapeutic development, tissue-specific genetic engineering, and genetic disease prediction will greatly benefit from this resource.

Codon and Codon-Pair Usage Tables (CoCoPUTs): Facilitating Genetic Variation Analyses and Recombinant Gene Design.

Usage of sequential codon-pairs is non-random and unique to each species. Codon-pair bias is related to but clearly distinct from individual codon usage bias. Codon-pair bias is thought to affect translational fidelity and efficiency and is presumed to be under the selective pressure. It was suggested that changes in codon-pair utilization may affect human disease more significantly than changes in single codons. Although recombinant gene technologies often take codon-pair usage bias into account, codon-pair usage data/tables are not readily available, thus potentially impeding research efforts. The present computational resource (https://hive.biochemistry.gwu.edu/review/codon2) systematically addresses this issue. Building on our recent HIVE-Codon Usage Tables, we constructed a new database to include genomic codon-pair and dinucleotide statistics of all organisms with sequenced genome, available in the GenBank. We believe that the growing understanding of the importance of codon-pair usage will make this resource an invaluable tool to many researchers in academia and pharmaceutical industry.

Effects of codon optimization on coagulation factor IX translation and structure: Implications for protein and gene therapies.

Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.