RESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/fisiología , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatina/genética , Simulación por Computador , Células HEK293 , Células HeLa , Humanos , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
Methylation of histone H3 lysine 36 (H3K36me) by yeast Set2 is critical for the maintenance of chromatin structure and transcriptional fidelity. However, we do not know the full range of Set2/H3K36me functions or the scope of mechanisms that regulate Set2-dependent H3K36 methylation. Here, we show that the APC/CCDC20 complex regulates Set2 protein abundance during the cell cycle. Significantly, absence of Set2-mediated H3K36me causes a loss of cell cycle control and pronounced defects in the transcriptional fidelity of cell cycle regulatory genes, a class of genes that are generally long, hence highly dependent on Set2/H3K36me for their transcriptional fidelity. Because APC/C also controls human SETD2, and SETD2 likewise regulates cell cycle progression, our data imply an evolutionarily conserved cell cycle function for Set2/SETD2 that may explain why recurrent mutations of SETD2 contribute to human disease.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclo Celular/genética , Regulación Fúngica de la Expresión Génica , Metiltransferasas/genética , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Evolución Biológica , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Ciclo Celular/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Metiltransferasas/metabolismo , Nocodazol/farmacología , Proteolisis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear ß-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.
Asunto(s)
Trastorno Autístico/metabolismo , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
Trypanosomes possess a unique mitochondrial genome called the kinetoplast DNA (kDNA). Many kDNA genes encode pre-mRNAs that must undergo guide RNA-directed editing. In addition, alternative mRNA editing gives rise to diverse mRNAs and several kDNA genes encode open reading frames of unknown function. To better understand the mechanism of RNA editing and the function of mitochondrial RNAs in trypanosomes, we have developed a reverse genetic approach using artificial site-specific RNA endonucleases (ASREs) to directly silence kDNA-encoded genes. The RNA-binding domain of an ASRE can be programmed to recognize unique 8-nucleotide sequences, allowing the design of ASREs to cleave any target RNA. Utilizing an ASRE containing a mitochondrial localization signal, we targeted the extensively edited mitochondrial mRNA for the subunit A6 of the F0F1 ATP synthase (A6) in the procyclic stage of Trypanosoma brucei. This developmental stage, found in the midgut of the insect vector, relies on mitochondrial oxidative phosphorylation for ATP production with A6 forming the critical proton half channel across the inner mitochondrial membrane. Expression of an A6-targeted ASRE in procyclic trypanosomes resulted in a 50% reduction in A6 mRNA levels after 24 h, a time-dependent decrease in mitochondrial membrane potential (ΔΨm), and growth arrest. Expression of the A6-ASRE, lacking the mitochondrial localization signal, showed no significant growth defect. The development of the A6-ASRE allowed the first in vivo functional analysis of an edited mitochondrial mRNA in T. brucei and provides a critical new tool to study mitochondrial RNA biology in trypanosomes.
Asunto(s)
Endonucleasas/metabolismo , Técnicas de Silenciamiento del Gen , ARN Protozoario/genética , ARN/genética , Trypanosoma brucei brucei/genética , Animales , Edición de ARN , ARN MitocondrialRESUMEN
Myotonic dystrophy type 1 (DM1) is caused by the expansion of (CTG)n in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, which is transcribed as (CUG)n repeats that accumulate in the nucleus. The RNA repeats specifically sequester or change the expression levels of several RNA-binding proteins, leading to aberrant splicing of many target genes. In this study, we developed artificial site-specific RNA endonucleases (ASREs) that specifically bind and cleave (CUG)n repeats RNA. We have generated one ASRE that can target the expanded RNA repeats in DM1 patient cells and specifically degrade the pathogenic DMPK messenger RNAs with minimal effect on wild-type alleles. Such ASRE treatment significantly decreased the number of nuclear foci in DM1 patient cells and can reverse the missplicing of many genes affected in DM1 patients. Taken together, the application of ASRE provides a new route of gene therapy for DM1 treatment.
Asunto(s)
Endorribonucleasas/metabolismo , Distrofia Miotónica/genética , Repeticiones de Trinucleótidos , Empalme Alternativo , Animales , Dominio Catalítico , Línea Celular , Endorribonucleasas/química , Endorribonucleasas/genética , Expresión Génica , Humanos , Hidrólisis , Distrofia Miotónica/enzimología , Distrofia Miotónica/terapia , Unión Proteica , Repeticiones de Trinucleótidos/genéticaRESUMEN
A large number of RNA-binding proteins play critical roles in controlling eukaryotic gene expression at multiple RNA-processing steps. Many of these proteins have modular configuration, containing a RNA binding domain to recognize their target and functional module to affect RNA metabolism. This simple configuration motivated the design of artificial factors that specifically manipulate RNA. While significant progress has been made since 1990s to engineer DNA binding proteins with designed specificity, design of analogous RNA binding factors was not practical until recently. With the increasing complexity of biological pathways involving RNA regulation, engineering RNA binding factors with customized specificity and function has become an emerging field of research. Such factors can serve as novel method to manipulate RNA metabolism and thus are very useful in basic biological and medical research. Here we discuss the current advances in engineering RNA binding proteins, with emphasis on the design principles and their potential applications as new therapeutic reagents and basic biological tools.
Asunto(s)
Ingeniería de Proteínas/métodos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN/genética , ARN/metabolismo , Animales , Humanos , ARN/química , Proteínas de Unión al ARN/químicaRESUMEN
NADP-Glutamate dehydrogenase (NADP-GDH) located at the interface of carbon and nitrogen metabolism has the potential to dictate fungal carbon flux. NADP-GDH from Aspergillus terreus, itaconate producer and an opportunistic pathogen, was purified to homogeneity using novel reactive dye-affinity resins. The pure enzyme was extensively characterized for its biochemical and kinetic properties and compared with its well studied Aspergillus niger counterpart. The A. terreus NADP-GDH was more stable and showed non-competitive ammonium inhibition with respect to glutamate. It exhibited hyperbolic 2-oxoglutarate saturation albeit with a weak substrate inhibition. This is in contrast to the allosteric nature of the enzyme from other Aspergilli. Differential susceptibility to chymotrypsin is also consistent with the absence of substrate cooperativity and conformational changes associated with A. terreus NADP-GDH. The non-allosteric nature of A. terreus NADP-GDH provides a unique opportunity to assess the contribution of allostery in metabolic regulation.
Asunto(s)
Aspergillus niger/enzimología , Aspergillus/enzimología , Proteínas Fúngicas/química , Glutamato Deshidrogenasa (NADP+)/química , Regulación Alostérica , Secuencia de Aminoácidos , Aspergillus/química , Aspergillus/genética , Aspergillus niger/química , Aspergillus niger/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Glutamato Deshidrogenasa (NADP+)/genética , Glutamato Deshidrogenasa (NADP+)/aislamiento & purificación , Glutamato Deshidrogenasa (NADP+)/metabolismo , Cinética , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
NADP-glutamate dehydrogenase (NADP-GDH) along with glutamine synthetase plays a pivotal role in ammonium assimilation. Specific inhibitors were valuable in defining the importance of glutamine synthetase in nitrogen metabolism. Selective in vivo inhibition of NADP-GDH has so far been an elusive desideratum. Isophthalate, a potent in vitro inhibitor of Aspergillus niger NADP-GDH [Noor S, Punekar NS. Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface. Microbiology 2005;151:1409-19], was evaluated for its efficacy in vivo. Dimethyl ester of isophthalate (DMIP), but not isophthalate, inhibited A. niger growth on agar as well as in liquid culture. This was ascribed to the inability of isophthalate to enter fungal mycelia. Subsequent to DMIP addition however, intracellular isophthalate could be demonstrated. Apart from NAD-GDH, no other enzyme including NAD-glutamate synthase was inhibited by isophthalate. A cross-over at NADP-GDH step of metabolism was observed as a direct consequence of isophthalate (formed in vivo from DMIP) inhibiting this enzyme. Addition of ammonium to DMIP-treated A. niger mycelia resulted in intensive vacuolation, retraction of cytoplasm and autolysis. Taken together, these results implicate glutamate dehydrogenase and NADP-GDH in particular, as a key target of in vivo isophthalate inhibition during ammonium assimilation.
RESUMEN
Irrespective of their pyridine nucleotide specificity, all glutamate dehydrogenases share a common chemical mechanism that involves an enzyme bound 'iminoglutarate' intermediate. Three compounds, structurally related to this intermediate, were tested for the inhibition of purified NADP-glutamate dehydrogenases from two Aspergilli, as also the bovine liver NAD(P)-glutamate dehydrogenase. 2-Methyleneglutarate, closely resembling iminoglutarate, was a potent competitive inhibitor of the glutamate dehydrogenase reaction. This is the first report of a non-aromatic structure with a better glutamate dehydrogenase inhibitory potency than aryl carboxylic acids such as isophthalate. A suitably located 2-methylene group to mimic the iminium ion could be exploited to design inhibitors of other amino acid dehydrogenases.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutaratos/farmacología , Ácidos Ftálicos/farmacología , Piridinas/farmacología , Animales , Aspergillus/enzimología , Aspergillus niger/enzimología , Catálisis/efectos de los fármacos , Bovinos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Glutamato Deshidrogenasa/metabolismo , Glutamatos/metabolismo , Glutaratos/química , Cinética , Hígado/enzimología , Estructura Molecular , NADP/metabolismoRESUMEN
The oncogenic transcription factor FoxM1 plays a vital role in cell cycle progression, is activated in numerous human malignancies, and is linked to chromosome instability. We characterize here a cullin 4-based E3 ubiquitin ligase and its substrate receptor, VprBP/DCAF1 (CRL4VprBP), which we show regulate FoxM1 ubiquitylation and degradation. Paradoxically, we also found that the substrate receptor VprBP is a potent FoxM1 activator. VprBP depletion reduces expression of FoxM1 target genes and impairs mitotic entry, whereas ectopic VprBP expression strongly activates a FoxM1 transcriptional reporter. VprBP binding to CRL4 is reduced during mitosis, and our data suggest that VprBP activation of FoxM1 is ligase independent. This implies a nonproteolytic activation mechanism that is reminiscent of, yet distinct from, the ubiquitin-dependent transactivation of the oncoprotein Myc by other E3s. Significantly, VprBP protein levels were upregulated in high-grade serous ovarian patient tumors, where the FoxM1 signature is amplified. These data suggest that FoxM1 abundance and activity are controlled by VprBP and highlight the functional repurposing of E3 ligase substrate receptors independent of the ubiquitin system.
Asunto(s)
Proteínas Portadoras/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Proteína Forkhead Box M1/metabolismo , Neoplasias Ováricas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Portadoras/genética , Ciclo Celular , Inestabilidad Cromosómica , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Femenino , Proteína Forkhead Box M1/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteínas Serina-Treonina Quinasas , Proteolisis , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The oncogenic AKT kinase is a key regulator of apoptosis, cell growth, and cell-cycle progression. Despite its important role in proliferation, it remains largely unknown how AKT is mechanistically linked to the cell cycle. We show here that cyclin F, a substrate receptor F-box protein for the SCF (Skp1/Cul1/F-box) family of E3 ubiquitin ligases, is a bona fide AKT substrate. Cyclin F expression oscillates throughout the cell cycle, a rare feature among the 69 human F-box proteins, and all of its known substrates are involved in proliferation. AKT phosphorylation of cyclin F enhances its stability and promotes assembly into productive E3 ligase complexes. Importantly, expression of mutant versions of cyclin F that cannot be phosphorylated by AKT impair cell-cycle entry. Our data suggest that cyclin F transmits mitogen signaling through AKT to the core cell-cycle machinery. This discovery has potential implications for proliferative control in malignancies where AKT is activated.
Asunto(s)
Ciclo Celular/fisiología , Ciclinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fosforilación , Transducción de SeñalRESUMEN
The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclinas/metabolismo , Retroalimentación Fisiológica/fisiología , Fase S/fisiología , Células HEK293 , Células HeLa , HumanosRESUMEN
NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical 'one-way' active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.
Asunto(s)
Aspergillus niger/enzimología , Cisteína/metabolismo , Disulfuros/metabolismo , Etanol/análogos & derivados , Glutamato Deshidrogenasa (NADP+)/metabolismo , Secuencia de Aminoácidos , Aspergillus niger/química , Dominio Catalítico , Cisteína/química , Desaminación , Etanol/metabolismo , Glutamato Deshidrogenasa (NADP+)/química , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Alineación de SecuenciaRESUMEN
Alternative splicing of pre-messenger RNA (mRNA) is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA-binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The carboxy-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk (extracellular signal-regulated protein kinase) pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq, we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk-regulated cell proliferation.
Asunto(s)
Empalme Alternativo/fisiología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Quinasas Quinasa Quinasa PAM/fisiología , Proteínas de Unión al ARN/fisiología , Células Cultivadas , Exones/fisiología , Células HEK293 , Ribonucleoproteínas Nucleares Heterogéneas/fisiología , Homeostasis/fisiología , Humanos , Riñón/citología , Riñón/fisiología , Fosforilación/fisiología , Transducción de Señal/fisiologíaRESUMEN
To better understand splicing regulation, we used a cell-based screen to identify ten diverse motifs that inhibit splicing from introns. Motifs were validated in another human cell type and gene context, and their presence correlated with in vivo splicing changes. All motifs exhibited exonic splicing enhancer or silencer activity, and grouping these motifs according to their distributions yielded clusters with distinct patterns of context-dependent activity. Candidate regulatory factors associated with each motif were identified, to recover 24 known and new splicing regulators. Specific domains in selected factors were sufficient to confer intronic-splicing-silencer activity. Many factors bound multiple distinct motifs with similar affinity, and all motifs were recognized by multiple factors, which revealed a complex overlapping network of protein-RNA interactions. This arrangement enables individual cis elements to function differently in distinct cellular contexts, depending on the spectrum of regulatory factors present.
Asunto(s)
Intrones , Empalme del ARN , Composición de Base , Línea Celular , Células HEK293 , Células HeLa , Humanos , Interferencia de ARN , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Specific cleavage of RNAs is critical for in vitro manipulation of RNA and for in vivo gene silencing. Here we engineer artificial site-specific RNA endonucleases to function analogously to DNA restriction enzymes. We combine a general RNA cleavage domain with a series of Pumilio/fem-3-binding factor domains that specifically recognize different 8-nucleotide RNA sequences. The resulting artificial site-specific RNA endonucleases specifically recognize RNA substrates and efficiently cleave near their binding sites. The artificial site-specific RNA endonucleases can be devised to recognize and cleave various RNA target sequences, providing a useful tool to manipulate RNAs in vitro. In addition, we generate designer artificial site-specific RNA endonucleases to specifically silence an endogenous gene in Escherichia coli, as well as a mitochondrial-encoded gene in human cells, suggesting that artificial site-specific RNA endonucleases can serve as a gene-silencing tool with designed specificity.
Asunto(s)
Endorribonucleasas/genética , Ingeniería de Proteínas/métodos , Sitios de Unión , Endorribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Silenciador del Gen , Genes Mitocondriales/efectos de los fármacos , Genes Mitocondriales/genética , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , División del ARNRESUMEN
Initial acceptance of Cibacron Blue 3G-A based matrices has made dye-ligand affinity chromatography an attractive proposition. This prompted the synthesis and search for new dye structures. A systematic library of 96 affinity resins was generated using novel analogs of Cibacron Blue 3G-A and also by varying spacer lengths for immobilization. The library was tested in a batch binding and elution mode using seven different proteins--four Aspergillus enzymes namely, NADP-glutamate dehydrogenase, laccase, glutamine synthetase and arginase, bovine pancreatic trypsin and the two serum proteins human serum albumin and immunoglobulin G. Unique binding patterns were observed for each of them indicating that the library displayed discriminatory interactions. The significance of spacer length in the interaction with proteins was discernable. Trypsin interacted best with affinity resins that had no spacer. It was possible to resolve IgG and HSA from a mixture using a combination of resins. There was a good spread of HSA binding capacity in the 96 affinity resins. While some showed better HSA binding capacity than the commercial CB3GA-based matrix, a few with lower capacity were also observed. Subsequent to an initial screen, one affinity resin (CR-017) could be used to enrich Aspergillus terreus NADP-GDH from crude cell extracts. The efficacy of this dye-affinity resin was rationalized by characterizing NADP-GDH inhibition kinetics with the corresponding free dye ligand. In the sum, the library provides a set of dye-ligand affinity matrices with a potential for use in high throughput screening for protein purification.