Cluster Of Amino Acids Research Articles

Microbiome-derived metabolites in early to mid-pregnancy and risk of gestational diabetes: a metabolome-wide association study

BackgroundPre-diagnostic disturbances in the microbiome-derived metabolome have been associated with an increased risk of diabetes in non-pregnant populations. However, the roles of microbiome-derived metabolites, the end-products of microbial metabolism, in gestational diabetes (GDM) remain understudied. We examined the prospective association of microbiome-derived metabolites in early to mid-pregnancy with GDM risk in a diverse population.MethodsWe conducted a prospective discovery and validation study, including a case–control sample of 91 GDM and 180 non-GDM individuals within the multi-racial/ethnic The Pregnancy Environment and Lifestyle Study (PETALS) as the discovery set, a random sample from the PETALS (42 GDM, 372 non-GDM) as validation set 1, and a case–control sample (35 GDM, 70 non-GDM) from the Gestational Weight Gain and Optimal Wellness randomized controlled trial as validation set 2. We measured untargeted fasting serum metabolomics at gestational weeks (GW) 10–13 and 16–19 by gas chromatography/time-of-flight mass spectrometry (TOF–MS), liquid chromatography (LC)/quadrupole TOF–MS, and hydrophilic interaction LC/quadrupole TOF–MS. GDM was diagnosed using the 3-h, 100-g oral glucose tolerance test according to the Carpenter-Coustan criteria around GW 24–28.ResultsAmong 1362 annotated compounds, we identified 140 of gut microbiome metabolism origin. Multivariate enrichment analysis illustrated that carbocyclic acids and branched-chain amino acid clusters at GW 10–13 and the unsaturated fatty acids cluster at GW 16–19 were positively associated with GDM risk (FDR < 0.05). At GW 10–13, the prediction model that combined conventional risk factors and LASSO-selected microbiome-derived metabolites significantly outperformed the model with only conventional risk factors including fasting glucose (discovery AUC: 0.884 vs. 0.691; validation 1: 0.945 vs. 0.731; validation 2: 0.987 vs. 0.717; all P < 0.01). At GW 16–19, similar results were observed (discovery AUC: 0.802 vs. 0.691, P < 0.01; validation 1: 0.826 vs. 0.780; P = 0.10).ConclusionsDysbiosis in microbiome-derived metabolites is present early in pregnancy among individuals progressing to GDM.

Identification and functional characterization of the nuclear and nucleolar localization signals in the intrinsically disordered region of nucleomethylin.

The nucleolar localization of proteins is regulated by specific signals directing their trafficking to nucleus and nucleolus. Here, we elucidate the mechanism underlying the nuclear and nucleolar localization of the nucleomethylin (NML) protein, focusing on its nuclear localization signals (NLSs) and nucleolar localization signal (NoLS). Using a combination of bioinformatic analysis and experimental validation, we identified two monopartite and one bipartite NLS motifs within NML. The combined presence of both monopartite NLSs significantly enhances nuclear localization of the protein, while specific basic amino acid clusters within the bipartite NLS are crucial for their functionality. We also reveal the functional role of the NLS-coupled NoLS motif in driving nucleolar localization of NML, which contains an arginine-rich motif essential for its function. The basic residues of the arginine-rich motif of NoLS of NML interacts with nucleophosmin 1 (NPM1), allowing the possible liquid-liquid phase separation and retention of NML in the nucleolus. Remarkably, the strong NoLS of NML can direct the nucleolar localization of a cytosolic protein, aldolase, emphasizing its potency. Overall, our findings provide insights into the combinatorial functioning of NLSs and NoLS in regulating the subcellular localization of NML, highlighting the intricate regulatory mechanisms governing its localization within the nucleus and nucleolus.

B cell epitope mapping: The journey to better vaccines and therapeutic antibodies

B-cell epitope mapping is an approach that can identify and characterise specific antigen binding sites of B-cell receptors and secreted antibodies. The ability to determine the antigenic clusters of amino acids bound by B-cell clones provides unprecedented detail that will aid in developing novel and effective vaccine targets and therapeutic antibodies for various diseases. Here, we discuss conventional approaches and emerging techniques that are used to map B-cell epitopes.

The spatial separation of basic amino acids is similar in RHAMM and hyaluronan binding peptide P15-1 despite different sequences and conformations.

Peptides that increase pro-reparative responses to injury and disease by modulating the functional organization of hyaluronan (HA) with its cell surface binding proteins (e.g., soluble receptor for hyaluronan-mediated motility [RHAMM] and integral membrane CD44) have potential therapeutic value. The binding of RHAMM to HA is an attractive target, since RHAMM is normally absent or expressed at low levels in homeostatic conditions, but its expression is significantly elevated in the extracellular matrix during tissue stress, response-to-injury, and in cancers and inflammation-based diseases. The HA-binding site in RHAMM contains two closely spaced sequences of clustered basic amino acids, in an alpha-helical conformation. In the present communication, we test whether an alpha-helical conformation is required for effective peptide binding to HA, and competitive disruption of HA-RHAMM interaction. The HA-binding RHAMM-competitive peptide P15-1, identified using the unbiased approach of phage display, was examined using circular dichroism spectroscopy and the conformation-predictive AI-based AlphaFold2 algorithm. Unlike the HA-binding site in RHAMM, peptide P15-1 was found to adopt irregular conformations in solution rather than alpha helices. Instead, our structural analysis suggests that the primary determinant of peptide-HA binding is associated with a specific clustering and spacing pattern of basic amino acids, allowing favorable electrostatic interaction with carboxylate groups on HA.

Diverse crystalline protein scaffolds through metal-dependent polymorphism.

As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co-crystallization with metal salts, exploiting the innate metal ion geometric requirements. A single structurally exposed negative amino acid cluster was responsible for metal coordination, despite the abundance of negatively charged residues. Density functional theory calculations showed that while most of the crystals were the most thermodynamically stable assemblies, some were kinetically trapped states. Thus, crystalline porosity diversity is achieved and controlled with metal coordination, opening a new scope in the application of proteins as biocompatible protein-metal-organic frameworks (POFs). In addition, metal-dependent polymorphic crystals allow direct comparison of metal coordination preferences.

Designing a structure-function alphabet of helix based on reduced amino acid clusters

Several simple secondary structures could form complex and diverse functional proteins, meaning that secondary structures may contain a lot of hidden information and are arranged according to certain principles, to carry enough information of functional specificity and diversity. However, these inner information and principles have not been understood systematically. In our study, we designed a structure-function alphabet of helix based on reduced amino acid clusters to describe the typical features of helices and delve into the information. Firstly, we selected 480 typical helices from membrane proteins, zymoproteins, transcription factors, and other proteins to define and calculate the interval range, and the helices are classified in terms of hydrophilicity, charge and length: (1) hydrophobic helix (≤43%), amphiphilic helix (43%∼71%), and hydrophilic helix (≥71%). (2) positive helix, negative helix, electrically neutral helix and uncharged helix. (3) short helix (≤8 aa), medium-length helix (9–28 aa), and long helix (≥29 aa). Then, we designed an alphabet containing 36 triplet codes according to the above classification, so that the main features of each helix can be represented by only three letters. This alphabet not only preliminarily defined the helix characteristics, but also greatly reduced the informational dimension of protein structure. Finally, we present an application example to demonstrate the value of the structure-function alphabet in protein functional determination and differentiation.

Rice bran peptide with α-glucosidase inhibition activity: Preparation, evaluation and molecular mechanism

The role of bioactive peptides in diabetes management has attracted extensive attention. In this study, the 1–3 kDa fraction of rice bran peptides (RBPs) was obtained by the hydrolysis of alkaline and dispase, and the following ultrafiltration demonstrated excellent α-glucosidase inhibition activity with an IC50 value of 1.17 ± 0.08 mg/mL and exhibited postprandial hypoglycemic activity in vivo. Enzymatic kinetics studies revealed that RBPs (1–3 kDa) inhibited α-glucosidase via competitive inhibition. Thirty-two peptides were characterized from RBPs (1–3 kDa) by LC-MS/MS. Virtual screening was applied to screening the new potential α-glucosidase inhibitor, and NVEKKDPTGA (NA-10) with a binding energy of −10.24 kcal/mol to α-glucosidase was selected as the most promising peptide. Molecular docking reflected that Ala at the C-terminal and KK amino acid cluster play a key role in forming hydrogen bonds with the catalytic site of α-glucosidase, which might be critical for the α-glucosidase inhibitory effect. This study proved that RBPs could be a promising bio-factor food ingredient for diabetes prevention.

Molecular recognition of niacin and lipid-lowering drugs by the human hydroxycarboxylic acid receptor 2

Niacin, an age-old lipid-lowering drug, acts through the hydroxycarboxylic acid receptor 2 (HCAR2), a G-protein-coupled receptor (GPCR). Yet, its use is hindered by side effects like skin flushing. To address this, specific HCAR2 agonists, like MK-6892 and GSK256073, with fewer adverse effects have been created. However, the activation mechanism of HCAR2 by niacin and these new agonists is not well understood. Here, we present three cryoelectron microscopy structures of Gi-coupled HCAR2 bound to niacin, MK-6892, and GSK256073. Our findings show that different ligands induce varying binding pockets in HCAR2, influenced by aromatic amino acid clusters (W91ECL1, H1614.59, W1885.38, H1895.39, and F1935.43) from receptors ECL1, TM4, and TM5. Additionally, conserved residues R1113.36 and Y2847.43, unique to the HCA receptor family, likely initiate activation signal propagation in HCAR2. This study provides insights into ligand recognition, receptor activation, and G protein coupling mediated by HCAR2, laying the groundwork for developing HCAR2-targeted drugs.

The intracellular C-terminal domain of mGluR6 contains ER retention motifs

Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.

Outer membrane protein OMP76 of Riemerella anatipestifer contributes to complement evasion and virulence by binding to duck complement factor vitronectin

ABSTRACT Riemerella anatipestifer is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin (Vn) is a complementary regulatory protein that inhibits the formation of the membrane attack complex (MAC). Microbes use outer membrane proteins (OMPs) to hijack Vn for complement evasion. However, the mechanism by which R. anatipestifer achieves evasion is unclear. This study aimed to characterise OMPs of R. anatipestifer which interact with duck Vn (dVn) during complement evasion. Far-western assays and comparison of wild-type and mutant strains that were treated with dVn and duck serum demonstrated particularly strong binding of OMP76 to dVn. These data were confirmed with Escherichia coli strains expressing and not expressing OMP76. Combining tertiary structure analysis and homology modelling, truncated and knocked-out fragments of OMP76 showed that a cluster of critical amino acids in an extracellular loop of OMP76 mediate the interaction with dVn. Moreover, binding of dVn to R. anatipestifer inhibited MAC deposition on the bacterial surface thereby enhancing survival in duck serum. Virulence of the mutant strain ΔOMP76 was attenuated significantly relative to the wild-type strain. Furthermore, adhesion and invasion abilities of ΔOMP76 decreased, and histopathological changes showed that ΔOMP76 was less virulent in ducklings. Thus, OMP76 is a key virulence factor of R. anatipestifer. The identification of OMP76-mediated evasion of complement by recruitment of dVn contributes significantly to the understanding of the molecular mechanism by which R. anatipestifer escapes host innate immunity and provides a new target for the development of subunit vaccines.

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

Sodium channel protein alpha subunit genomics: building an integrated knowledgebase to translate between family members

The sodium channel alpha proteins consist of ten homologous human proteins (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A, SCN11A), many involved in various human physiological pathways and clinical disorders/syndromes. Many human variants connected to these genes remain uncertain for human disease. To better support the understanding of challenging human variants, we have built an ortholog database of the alpha subunit proteins for the sodium channel. We combined 1,680 vertebrate species sequences of family members, protein modeling, molecular dynamic simulation of lipid embedded protein, screening of human variants (gnomAD, ClinVar, Geno2MP), and analysis of a large number of human RNAseq and single-cell RNA-seq to understand the function of each amino acid in the human proteins and how the amino acids are conserved across family members to contribute to sodium channel functions within diverse cells of human. There are 610 amino acids conserved across the alpha subunit family, representing the most conserved amino acids for each member. These amino acids drive the structural fold, membrane interactions, and channel opening. A cluster of amino acids is conserved between a subset of family members that defines subclusters of alpha subunit proteins. Expression analysis of each member reveals clusters of members co-expressed in various cells, where heart and brain datasets show the specificity of cell types that contribute to genotype-to-phenotype associations of each member. Further, to support variant characterizations, the ortholog alignment data provides a unique ability to discover loss vs. gain of function variants found between members, allowing for known pathogenic variants of one member to aid in the characterization of another member's uncertain variants. For example, a clinical variant of SCN8A linked to seizures was supported as loss-of-function through variants within SCN1A (epilepsy), SCN2A (seizures), SCN4A (Familial hyperkalemic periodic paralysis), and SCN5A (Brugada syndrome). Overall, we show an exciting and promising new tool for ortholog genomic mapping that can support clinical variant insights into physiological outcomes (genotype-to-phenotype relationship), which should be applied to other cellular ion channels and transporters. This research was funded by the Gerber Foundation (to JWP), National Institutes of Health (K01ES025435 to JWP), and Michigan State University. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of Myelin Basic Protein Proximity Interactome Using TurboID Labeling Proteomics.

Myelin basic protein (MBP) is one of the key structural elements of the myelin sheath and has autoantigenic properties in multiple sclerosis (MS). Its intracellular interaction network is still partially deconvoluted due to the unfolded structure, abnormally basic charge, and specific cellular localization. Here we used the fusion protein of MBP with TurboID, an engineered biotin ligase that uses ATP to convert biotin to reactive biotin-AMP that covalently attaches to nearby proteins, to determine MBP interactome. Despite evident benefits, the proximity labeling proteomics technique generates high background noise, especially in the case of proteins tending to semi-specific interactions. In order to recognize unique MBP partners, we additionally mapped protein interaction networks for deaminated MBP variant and cyclin-dependent kinase inhibitor 1 (p21), mimicking MBP in terms of natively unfolded state, size and basic amino acid clusters. We found that in the plasma membrane region, MBP is colocalized with adhesion proteins occludin and myelin protein zero-like protein 1, solute carrier family transporters ZIP6 and SNAT1, Eph receptors ligand Ephrin-B1, and structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3), protein transport protein hSec23B and cytoplasmic dynein 1 heavy chain 1. We also detected that MBP potentially interacts with proteins involved in Fe2+ and lipid metabolism, namely, ganglioside GM2 activator protein, long-chain-fatty-acid-CoA ligase 4 (ACSL4), NADH-cytochrome b5 reductase 1 (CYB5R1) and metalloreductase STEAP3. Assuming the emerging role of ferroptosis and vesicle cargo docking in the development of autoimmune neurodegeneration, MBP may recruit and regulate the activity of these processes, thus, having a more inclusive role in the integrity of the myelin sheath.

Peptide Bond Formation in the Protonated Serine Dimer Following Vacuum UV Photon‐Induced Excitation

AbstractPossible routes for intra‐cluster bond formation (ICBF) in protonated serine dimers have been studied. We found no evidence of ICBF following low energy collision‐induced dissociation (in correspondence with previous works), however, we do observe clear evidence for ICBF following photon absorption in the 4.6–14 eV range. Moreover, the comparison of photon‐induced dissociation measurements of the protonated serine dimer to those of a protonated serine dipeptide provides evidence that ICBF, in this case, involves peptide bond formation (PBF). The experimental results are supported by ab initio molecular dynamics and exploration of several excited state potential energy surfaces, unraveling a pathway for PBF following photon absorption. The combination of experiments and theory provides insight into the PBF mechanisms in clusters of amino acids, and reveals the importance of electronic excited states reached upon UV/VUV light excitation.

Peptide Bond Formation in the Protonated Serine Dimer Following Vacuum UV Photon-Induced Excitation.

Possible routes for intra-cluster bond formation (ICBF) in protonated serine dimers have been studied. We found no evidence of ICBF following low energy collision-induced dissociation (in correspondence with previous works), however, we do observe clear evidence for ICBF following photon absorption in the 4.6-14 eV range. Moreover, the comparison of photon-induced dissociation measurements of the protonated serine dimer to those of a protonated serine dipeptide provides evidence that ICBF, in this case, involves peptide bond formation (PBF). The experimental results are supported by ab initio molecular dynamics and exploration of several excited state potential energy surfaces, unraveling a pathway for PBF following photon absorption. The combination of experiments and theory provides insight into the PBF mechanisms in clusters of amino acids, and reveals the importance of electronic excited states reached upon UV/VUV light excitation.

Functional analysis of PsbS transmembrane domains through base editing in Physcomitrium patens.

Plants exposed to light fluctuations are protected from photodamage by non-photochemical quenching (NPQ), a reversible mechanism that enables dissipation of excess absorbed energy as heat, which is essential for plant fitness and crop productivity. In plants NPQ requires the presence of the membrane protein PsbS, which upon activation interacts with antenna proteins, inducing their dissipative conformation. Here, we exploited base editing (BE) in the moss Physcomitrium patens to introduce specific amino acid changes invivo and assess their impact on PsbS activity, targeting transmembrane regions to investigate their role in essential protein-protein interactions. This approach enabled the recognition of residues essential for protein stability and the identification of a hydrophobic cluster of amino acids impacting PsbS activity. This work provides new information on the molecular mechanism of PsbS while also demonstrating the potential of BE approaches for in planta gene function analysis.

Identification of the Actin-Binding Region and Binding to Host Plant Apple Actin of Immunodominant Transmembrane Protein of 'Candidatus Phytoplasma mali'.

'Candidatus Phytoplasma mali' ('Ca. P. mali') has only one major membrane protein, the immunodominant membrane protein (Imp), which is regarded as being close to the ancestor of all phytoplasma immunodominant membrane proteins. Imp binds to actin and possibly facilitates its movement in the plant or insect host cells. However, protein sequences of Imp are quite diverse among phytoplasma species, thus resulting in difficulties in identifying conserved domains across species. In this work, we compare Imp protein sequences of 'Ca. P. mali' strain PM19 (Imp-PM19) with Imp of different strains of 'Ca. P. mali' and identify its actin-binding domain. Moreover, we show that Imp binds to the actin of apple (Malus x domestica), which is the host plant of 'Ca. P. mali'. Using molecular and scanning force spectroscopy analysis, we find that the actin-binding domain of Imp-PM19 contains a highly positively charged amino acid cluster. Our result could allow investigating a possible correlation between Imp variants and the infectivity of the corresponding 'Ca. P. mali' isolates.

A coarse-grained molecular dynamics investigation on spontaneous binding of Aβ1–40 fibrils with cholesterol-mixed DPPC bilayers

Alzheimer's disease is the most common form of dementia. Its aetiology is characterized by the misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet-rich Aβ oligomers/fibrils. Although multiple experimental studies have suggested that Aβ oligomers/fibrils interact with the cell membranes and perturb their structures and dynamics, the molecular mechanism of this interaction is still not fully understood. In the present work, we have performed a total of 120 μs-long simulations to investigate the interaction between trimeric or hexameric Aβ1–40 fibrils with either a 100% DPPC bilayer, a 70% DPPC-30% cholesterol bilayer or a 50% DPPC-50% cholesterol bilayer. Our simulation data capture the spontaneous binding of the aqueous Aβ1–40 fibrils with the membranes and show that the central hydrophobic amino acid cluster, the lysine residue adjacent to it and the C-terminal hydrophobic residues are all involved in the process. Moreover, our data show that while the Aβ1–40 fibril does not bind to the 100% DPPC bilayer, its binding affinity for the membrane increases with the amount of cholesterol. Overall, our data suggest that two clusters of hydrophobic residues and one lysine help Aβ1–40 fibrils establish stable interactions with a cholesterol-rich DPPC bilayer. These residues are likely to represent potential target regions for the design of inhibitors, thus opening new avenues in structure-based drug design against Aβ oligomer/fibril-membrane interaction.

Selective confinement of potassium, rubidium, or caesium ions in a non-covalent hydroxyproline octamer cage stabilized by cis-hydroxyl locks.

While numerous studies have focused on the impact of chirality on some magic amino acid clusters, this article investigates the effects of steric isomerization using 4-hydroxyproline octamers as a model system. Through mass spectrometry, infrared photodissociation spectroscopy, and theoretical calculation, it was demonstrated that the cis-4-hydroxy-L-proline octamer can selectively cage potassium, rubidium, or caesium ions through stable cis-hydroxyl locks, while the trans-form cannot. The results highlight the importance of hydroxyl group orientation in designing biocompatible membrane transporters with high ion-selectivity.

Yeast Protein Asf1 Possesses Modulating Activity towards Protein Kinase CK2

Protein kinase CK2 plays an important role in cell survival and protects regulatory proteins from caspase-mediated degradation during apoptosis. The consensus sequence of proteins phosphorylated by CK2 contains a cluster of acidic amino acids around the phosphorylation site. The poly-acidic sequence in yeast protein Asf1 is similar to the acidic loop in CK2β, which possesses a regulatory function. We observed that the overexpression of Asf1 in yeast cells influences cell growth. Experiments performed in vitro and in vivo indicate that yeast protein Asf1 inhibits protein kinase CK2. Our data suggest that each CK2 isoform might be regulated in a different way. Deletion of the amino or carboxyl end of Asf1 reveals that the acidic cluster close to the C-terminus is responsible for the activation or inhibition of CK2 activity.