The F-box E3 ligase protein FBXO11 regulates EBNA3C-associated degradation of BCL6.

Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.

A minimal presynaptic protein machinery mediating synchronous and asynchronous exocytosis and short-term plasticity.

Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca2+ influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca2+-triggered vesicle fusion and short-term facilitation. We find that Synaptotagmin-1, Synaptotagmin-7, and Complexin, synergistically restrain SNARE complex assembly, thus preserving vesicles in a stably docked state at rest. Upon Ca2+ activation, Synaptotagmin-1 induces rapid vesicle fusion, while Synaptotagmin-7 mediates delayed fusion. Competitive binding of Synaptotagmin-1 and Synaptotagmin-7 to the same SNAREs, coupled with differential rates of Ca2+-triggered fusion clamp reversal, govern the kinetics of vesicular fusion. Under conditions mimicking sustained neuronal activity, the Synaptotagmin-7 fusion clamp is destabilized by the elevated basal Ca2+ concentration, thereby enhancing the synchronous component of fusion. These findings provide a direct demonstration that a small set of proteins is sufficient to account for how nerve terminals adapt and regulate the Ca2+-evoked neurotransmitter exocytosis process to support their specialized functions in the nervous system.

KSHV-encoded LANA bypasses transcriptional block through the stabilization of RNA Pol II in hypoxia.

IMPORTANCE: Hypoxia can induce the reactivation of Kaposi sarcoma-associated virus (KSHV), which necessitates the synthesis of critical structural proteins. Despite the unfavorable energetic conditions of hypoxia, KSHV utilizes mechanisms to prevent the degradation of essential cellular machinery required for successful reactivation. Our study provides new insights on strategies employed by KSHV-infected cells to maintain steady-state transcription by overcoming hypoxia-mediated metabolic stress to enable successful reactivation. Our discovery that the interaction of latency-associated nuclear antigen with HIF1α and NEDD4 inhibits its polyubiquitination activity, which blocks the degradation of RNA Pol II during hypoxia, is a significant contribution to our understanding of KSHV biology. This newfound knowledge provides new leads in the development of novel therapies for KSHV-associated diseases.

Attenuation of IFN signaling due to m6A modification of the host epitranscriptome promotes EBV lytic reactivation.

BACKGROUND: Reactivation of Epstein Barr virus (EBV) leads to modulation of the viral and cellular epitranscriptome. N6-methyladenosine (m6A) modification is a type of RNA modification that regulates metabolism of mRNAs. Previous reports demonstrated that m6A modification affects the stability and metabolism of EBV encoded mRNAs. However, the effect of reactivation on reprograming of the cellular mRNAs, and how this contributes to successful induction of lytic reactivation is not known. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptomic RNA sequencing (RNA-seq) and RNA pull-down PCR were used to screen and validate differentially methylated targets. Western blotting, quantitative real-time PCR (RT-qPCR) and immunocytochemistry were used to investigate the expression and localization of different proteins. RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of downstream genes. Insertion of point mutation to disrupt the m6A methylation sites was used to verify the effect of m6A methylation on its stability and expression levels. RESULTS: We report that during EBV reactivation the m6A eraser ALKBH5 is significantly downregulated leading to enhanced methylation of the cellular transcripts DTX4 and TYK2, that results in degradation of TYK2 mRNAs and higher efficiency of translation of DTX4 mRNAs. This resulted in attenuation of IFN signaling that promoted progression of viral lytic replication. Furthermore, inhibition of m6A methylation of these transcripts led to increased production of IFN, and a substantial reduction in viral copy number, which suggests abrogation of lytic viral replication. CONCLUSION: Our findings illuminate the significance of m6A modification in overcoming the innate immune response during EBV reactivation. We now report that during lytic reactivation EBV targets the RNA methylation system of the host to attenuate the innate immune response by suppressing the interferon signaling which facilitates successful lytic replication of the virus.

Spectrin: an alternate target for cytoskeletal drugs.

Cytoskeletal drugs having enormous therapeutic potential act on the cytoskeletal components like actin, tubulin either by promoting polymerization or destabilizing the same. Here we present the interaction of the popular cytoskeletal drugs such as taxol, latrunculin and cytochalasin with spectrin, a huge protein with multi domains that forms the cytoskeletal network. Particularly, the actin binding domain of spectrin regulates the dynamics of the actin cytoskeleton. We followed the binding of these drugs to its actin binding domain and intact spectrin as well. These drugs bind with moderate affinity (Kb ∼ 104 M-1) and the interaction with actin binding domain is entropy driven and hydrophobic in nature as determined by Van't Hoff plot. The docking studies and molecular dynamics simulations further corroborate the experimental findings. Particularly the higher binding constants in the case of latrunculin and cytochalasin to the actin binding domain of spectrin suggest the binding sites are presumably located in its actin binding domain.Communicated by Ramaswamy H. Sarma.

Structural Flexibility of Proteins Dramatically Alters Membrane Stability─A Novel Aspect of Lipid-Protein Interaction.

Protein isoforms are structural variants with changes in the overall flexibility predominantly at the tertiary level. For membrane associated proteins, such structural flexibility or rigidity affects membrane stability by playing modulatory roles in lipid-protein interaction. Herein, we investigate the protein chain flexibility mediated changes in the mechanistic behavior of phospholipid model membranes in the presence of two well-known isoforms, erythroid (ER) and nonerythroid (NER) spectrin. We show dramatic alterations of membrane elasticity and stability induced by spectrin in the Langmuir monolayers of phosphatidylocholine (PC) and phosphatidylethanolamine (PE) by a combination of isobaric relaxation, surface pressure-area isotherm, X-ray scattering, and microscopy measurements. The NER spectrin drives all monolayers to possess an approximately equal stability, and that required 25-fold increase and 5-fold decrease of stability in PC and PE monolayers, respectively. The untilting transition of the PC membrane in the presence of NER spectrin observed in X-ray measurements can explain better membrane packing and stability.

Epigenetic Reprogramming of Kaposi's Sarcoma-Associated Herpesvirus during Hypoxic Reactivation.

The biphasic life cycle (latent and lytic) of Kaposi's sarcoma-associated Herpesvirus (KSHV) is regulated by epigenetic modification of its genome and its associated histone proteins. The temporal events driving epigenetic reprogramming of the KSHV genome on initial infection to establish latency has been well studied, but the reversal of these epigenetic changes during lytic replication, especially under physiological conditions such as hypoxia, has not been explored. In this study, we investigated epigenetic reprogramming of the KSHV genome during hypoxic reactivation. Hypoxia induced extensive enrichment of both transcriptional activators and repressors on the KSHV genome through H3K4Me3, H3K9Me3, and H3K27Me3, as well as histone acetylation (H3Ac) modifications. In contrast to uniform quantitative enrichment with modified histones, a distinct pattern of RTA and LANA enrichment was observed on the KSHV genome. The enrichment of modified histone proteins was due to their overall higher expression levels, which was exclusively seen in KSHV-positive cells. Multiple KSHV-encoded factors such as LANA, RTA, and vGPCR are involved in the upregulation of these modified histones. Analysis of ChIP-sequencing for the initiator DNA polymerase (DNAPol1α) combined with single molecule analysis of replicated DNA (SMARD) demonstrated the involvement of specific KSHV genomic regions that initiate replication in hypoxia.

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins.

Hemoglobin oxidation due to oxidative stress and disease conditions leads to the generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the red blood cell (RBC) membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2', 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-ß-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

Comparative Analysis of Tryptophan Dynamics in Spectrin and Its Constituent Domains: Insights from Fluorescence.

Spectrin is a cytoskeletal protein ubiquitous in metazoan cells that acts as a liaison between the plasma membrane and the cellular interior and imparts mechanical stability to the plasma membrane. Spectrin is known to be highly dynamic, with an appreciable degree of torsional and segmental mobility. In this context, we have earlier utilized the red edge excitation shift (REES) approach to report the retention of restricted solvation dynamics and local structure in the vicinity of spectrin tryptophans on urea denaturation and loss of spectrin secondary structure. As a natural progression of our earlier work, in this work, we carried out a biophysical dissection of tryptophan solvation and rotational dynamics in spectrin and its constituent domains, in order to trace the origin of local structure retention observed in denatured spectrin. Our results show that the ankyrin binding domain (and, to a lesser extent, the ß-tetramerization domain) is capable of retention of local structure, similar to that observed for intact spectrin. However, all α-chain domains studied exhibit negligible retention of local structure on urea denaturation. Such a stark chain-specific retention of local structure could originate from the fact that the ß-chain domains possess specialized functions, where conservation of local (structural) integrity may be a prerequisite for optimum cellular function. To the best of our knowledge, these observations represent one of the first systematic biophysical dissections of spectrin dynamics in terms of its constituent domains and add to emerging literature on comprehensive domain-based analysis of spectrin organization, dynamics, and function.

Leishmania donovani infection induce Extracellular signal-regulated kinase ½ (ERK½) mediated lipid droplet generation in macrophages.

Recently unfolded mechanisms showed lipid droplet helps in pathogen survival and paralyzes host immune response. In the present study, we showed the extent of lipid droplet(LD) generation in Leishmania donovani infection, the signaling involved, and their function concerning pathogenicity. RAW 264.7 and J774A.1 cells were used to infect with L. donovani and then flow cytometry and confocal microscopy were used to detect lipid droplet generation and subsequent assays. In this study, we showed that L. donovani AG83 (AG83/MHOM/1983) triggers lipid droplet formation in macrophages in a time-dependent manner. We provide novel insight into the signaling molecules which is responsible for LD accumulation. Interestingly, LPG deficient attenuated Leishmania strain UR6 (UR6/MHOM/1978) failed to fuel LD generation. But inhibition of phagosome maturation drastically stimulates LD accumulation in UR6 infected MΦs. Aspirin treatment in AG83 infected MΦs does not only lower LD load but also favors phagolysosome biogenesis and corrects cytokine balance. Employing strategies to circumvent halt in phagosome maturation using drugs that manipulate lipid droplet generation could be used as a therapeutic tool to resist parasite growth in the early hour of infection.

Thermodynamics of adsorption of alcohol dehydrogenase on the gold nanoparticle surface: a model based analysis versus direct measurement.

Characterization of the nanoparticle protein corona has gained tremendous importance lately. The parameters which quantitatively establish a specific nanoparticle-protein interaction need to be measured accurately since good quality data are necessary for the elucidation of the underlying mechanism and accurate molecular dynamics simulation. Here, we have employed surface sensitive second harmonic light scattering (SHLS) for investigating the adsorption of a tetrameric protein, alcohol dehydrogenase (ADH, Saccharomyces cerevisiae 147 kDa), on 16 nm, 27 nm, 41 nm, and 69 nm citrate capped gold nanoparticles (GNPs) in aqueous phosphate buffer at pH 7. We have extracted the binding constant, number of ADH bound per GNP, Gibbs free energy (ΔG°) from the decay of the second harmonic scattered signal as a function of protein concentration using a modified version of the Langmuir adsorption isotherm. The data obtained were checked with another technique, dynamic light scattering, using the same modified Langmuir model (MLM). While the binding constants measured by the two methods are in agreement, the number of ADH bound to each GNP obtained by the two methods varies a lot. In order to further probe this binding independent of a model fitting, we used an orthogonal fluorescence assay which measures the number of ADH bound to a GNP directly, and no model-fitting is necessary. We then used temperature dependent SHLS to measure the heat of adsorption (ΔH°) and entropy (ΔS°) for ADH-GNP corona formation. We found that the equilibrium binding constant (Kb) obtained from SHLS is of the order of 109 M-1 and the formation of the GNP-ADH corona is spontaneous with ΔG° ∼ -55 kJ mol-1. However, the adsorption is modestly endothermic, accompanied by a large increase in entropy. Stated differently, GNP-ADH corona formation is entropically driven. This is perhaps due to the tremendous disruption of the water structure at the negatively charged interface upon the arrival of the protein within the bonding distance to it. We believe that the SHLS technique is highly sensitive and reliable, at very low concentrations of both nanoparticles and proteins, for the quantitative estimation of the thermodynamic parameters of nanoparticle-protein corona formation, where many other techniques may fall short.

Prognostic correlations with the microbiome of breast cancer subtypes.

Alterations to the natural microbiome are linked to different diseases, and the presence or absence of specific microbes is directly related to disease outcomes. We performed a comprehensive analysis with unique cohorts of the four subtypes of breast cancer (BC) characterized by their microbial signatures, using a pan-pathogen microarray strategy. The signature (includes viruses, bacteria, fungi, and parasites) of each tumor subtype was correlated with clinical data to identify microbes with prognostic potential. The subtypes of BC had specific viromes and microbiomes, with ER+ and TN tumors showing the most and least diverse microbiome, respectively. The specific microbial signatures allowed discrimination between different BC subtypes. Furthermore, we demonstrated correlations between the presence and absence of specific microbes in BC subtypes with the clinical outcomes. This study provides a comprehensive map of the oncobiome of BC subtypes, with insights into disease prognosis that can be critical for precision therapeutic intervention strategies.

KSHV-encoded vCyclin can modulate HIF1α levels to promote DNA replication in hypoxia.

The cellular adaptive response to hypoxia, mediated by high HIF1α levels includes metabolic reprogramming, restricted DNA replication and cell division. In contrast to healthy cells, the genome of cancer cells, and Kaposi's sarcoma associated herpesvirus (KSHV) infected cells maintains replication in hypoxia. We show that KSHV infection, despite promoting expression of HIF1α in normoxia, can also restrict transcriptional activity, and promoted its degradation in hypoxia. KSHV-encoded vCyclin, expressed in hypoxia, mediated HIF1α cytosolic translocation, and its degradation through a non-canonical lysosomal pathway. Attenuation of HIF1α levels by vCyclin allowed cells to bypass the block to DNA replication and cell proliferation in hypoxia. These results demonstrated that KSHV utilizes a unique strategy to balance HIF1α levels to overcome replication arrest and induction of the oncogenic phenotype, which are dependent on the levels of oxygen in the microenvironment.

HIF1α-Regulated Expression of the Fatty Acid Binding Protein Family Is Important for Hypoxic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

The hypoxic microenvironment and metabolic reprogramming are two major contributors to the phenotype of oncogenic virus-infected cells. Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) stabilizes hypoxia-inducible factor 1α (HIF1α) and reprograms cellular metabolism. We investigated the comparative transcriptional regulation of all major genes involved in fatty acid and amino acid metabolism in KSHV-positive and -negative cells grown under normoxic or hypoxic conditions. We show a distinct regulation of genes involved in both fatty acid and amino acid metabolism in KSHV-positive cells grown in either normoxic or hypoxic conditions, with a particular focus on genes involved in the acetyl coenzyme A (acetyl-CoA) pathway. The fatty acid binding protein (FABP) family of genes, specifically FABP1, FABP4, and FABP7, was also observed to be synergistically upregulated in hypoxia by KSHV. This pattern of FABP gene expression was also seen in naturally infected KSHV BC3 or BCBL1 cells when compared to KSHV-negative DG75 or BL41 cells. Two KSHV-encoded antigens, which positively regulate HIF1α, the viral G-protein coupled receptor (vGPCR), and the latency-associated nuclear antigen (LANA) were shown to drive upregulation of the FABP gene transcripts. Suppression of FABPs by RNA interference resulted in an adverse effect on hypoxia-dependent viral reactivation. Overall, this study provides new evidence, which supports a rationale for the inhibition of FABPs in KSHV-positive cells as potential strategies, for the development of therapeutic approaches targeting KSHV-associated malignancies.IMPORTANCE Hypoxia is a detrimental stress to eukaryotes and inhibits several cellular processes, such as DNA replication, transcription, translation, and metabolism. Interestingly, the genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to undergo productive replication in hypoxia. We investigated the comparative transcriptional regulation of all major genes involved in fatty acid and amino acid metabolism in KSHV-positive and -negative cells grown under normoxic or hypoxic conditions. Several metabolic pathways were observed differentially regulated by KSHV in hypoxia, specifically, the fatty acid binding protein (FABP) family genes (FABP1, FABP4, and FABP7). KSHV-encoded antigens, vGPCR and LANA, were shown to drive upregulation of the FABP transcripts. Suppression of FABPs by RNA interference resulted in an adverse effect on hypoxia-dependent viral reactivation. Overall, this study provides new evidence, which supports a rationale for the inhibition of FABPs in KSHV-positive cells as potential strategies, for the development of therapeutic approaches targeting KSHV-associated malignancies.

Detection of Microbial Agents in Oropharyngeal and Nasopharyngeal Samples of SARS-CoV-2 Patients.

The novel coronavirus outbreak started in December 2019 and rapidly spread around the globe, leading to a global pandemic. Here we reported the association of microbial agents identified in oropharyngeal and nasopharyngeal samples from patients with SARS-CoV-2 infection, using a Pan-microarray based technology referred to as PathoChIP. To validate the efficiency of PathoChIP, reference viral genomes obtained from BEI resource and 25 SARS-CoV-2 positive clinical samples were tested. This technology successfully detected femtogram levels of SARS-CoV-2 viral RNA, which demonstrated greater sensitivity and specificity than conventional diagnostic techniques. Simultaneously, a broad range of other microorganisms, including other viruses, bacteria, fungi and parasites can be detected in those samples. We identified 7 viral, 12 bacterial and 6 fungal agents common across all clinical samples suggesting an associated microbial signature in individuals who are infected with SARS-CoV-2. This technology is robust and has a flexible detection methodology that can be employed to detect the presence of all human respiratory pathogens in different sample preparations with precision. It will be important for differentiating the causative agents of respiratory illnesses, including SARS-CoV-2.

Vascular endothelial growth factor encoded by Parapoxviruses can regulate metabolism and survival of triple negative breast cancer cells.

Dysbiotic microbiomes are linked to many pathological outcomes including different metabolic disorders like diabetes, atherosclerosis and even cancer. Breast cancer is the second leading cause of cancer associated death in women, and triple negative breast cancer (TNBC) is the most aggressive type with major challenges for intervention. Previous reports suggested that Parapoxvirus signatures are one of the predominant dysbiotic viral signatures in TNBC. These viruses encode several genes that are homologs of human genes. In this study, we show that the VEGF homolog encoded by Parapoxviruses, can induce cell proliferation, and alter metabolism of breast cancer and normal breast cells, through alteration of MAPK-ERK and PI3K-AKT signaling. In addition, the activity of the transcription factor FoxO1 was altered by viral-encoded VEGF through activation of the PI3K-AKT pathway, leading to reprogramming of cellular metabolic gene expression. Therefore, this study provides new insights into the function of viral-encoded VEGFs, which promoted the growth of the breast cancer cells and imparted proliferative phenotype with altered metabolism in normal breast cells.

Multiple Functions of Spectrin: Convergent Effects.

Spectrin is a multifunctional, multi-domain protein most well known in the membrane skeleton of mature human erythrocytes. Here we review the literature on the crosstalk of the chaperone activity of spectrin with its other functionalities. We hypothesize that the chaperone activity is derived from the surface exposed hydrophobic patches present in individual "spectrin-repeat" domains and show a competition between the membrane phospholipid binding functionality and chaperone activity of spectrin. Moreover, we show that post-translational modifications such as glycation which shield these surface exposed hydrophobic patches, reduce the chaperone function. On the other hand, oligomerization which is linked to increase of hydrophobicity is seen to increase it. We note that spectrin seems to prefer haemoglobin as its chaperone client, binding with it preferentially over other denatured proteins. Spectrin is also known to interact with unstable haemoglobin variants with a higher affinity than in the case of normal haemoglobin. We propose that chaperone activity of spectrin could be important in the cellular biochemistry of haemoglobin, particularly in the context of diseases.

A Tripartite Interaction among the Basidiomycete Rhodotorula mucilaginosa, N2-Fixing Endobacteria, and Rice Improves Plant Nitrogen Nutrition.

Nitrogen (N) limits crop yield, and improvement of N nutrition remains a key goal for crop research; one approach to improve N nutrition is identifying plant-interacting, N2-fixing microbes. Rhodotorula mucilaginosa JGTA-S1 is a basidiomycetous yeast endophyte of narrowleaf cattail (Typha angustifolia). JGTA-S1 could not convert nitrate or nitrite to ammonium but harbors diazotrophic (N2-fixing) endobacteria (Pseudomonas stutzeri) that allow JGTA-S1 to fix N2 and grow in a N-free environment; moreover, P. stutzeri dinitrogen reductase was transcribed in JGTA-S1 even under adequate N. Endobacteria-deficient JGTA-S1 had reduced fitness, which was restored by reintroducing P. stutzeri JGTA-S1 colonizes rice (Oryza sativa), significantly improving its growth, N content, and relative N-use efficiency. Endofungal P. stutzeri plays a significant role in increasing the biomass and ammonium content of rice treated with JGTA-S1; also, JGTA-S1 has better N2-fixing ability than free-living P. stutzeri and provides fixed N to the plant. Genes involved in N metabolism, N transporters, and NODULE INCEPTION-like transcription factors were upregulated in rice roots within 24 h of JGTA-S1 treatment. In association with rice, JGTA-S1 has a filamentous phase and P. stutzeri only penetrated filamentous JGTA-S1. Together, these results demonstrate an interkingdom interaction that improves rice N nutrition.

Chaperone potential of erythroid spectrin: Effects of hemoglobin interaction, macromolecular crowders, phosphorylation and glycation.

Spectrin, the major protein component of the erythrocyte membrane skeleton has chaperone like activity and is known to bind membrane phospholipids and hemoglobin. We have probed the chaperone activity of spectrin in presence of hemoglobin and phospholipid SUVs of different compositions to elucidate the effect of phospholipid/hemoglobin binding on chaperone function. It is seen that spectrin displays a preference for hemoglobin over other substrates leading to a decrease in chaperone activity in presence of hemoglobin. A competition is seen to exist between phospholipid binding and chaperone function of spectrin, in a dose dependent manner with the greatest extent of decrease being seen in case of phospholipid vesicles containing aminophospholipids e.g. PS and PE which may have implications in diseases like hereditary spherocytosis where mutation in spectrin is implicated in its detachment from cell membrane. To gain a clearer understanding of the chaperone like activity of spectrin under in-vivo like conditions we have investigated the effect of macromolecular crowders as well as phosphorylation and glycation states on chaperone activity. It is seen that the presence of non-specific, protein and non-protein macromolecular crowders do not appreciably affect chaperone function. Phosphorylation also does not affect the chaperone function unlike glycation which progressively diminishes chaperone activity. We propose a model where chaperone clients adsorb onto spectrin's surface and processes that bind to and occlude these surfaces decrease chaperone activity.

Localizing the chaperone activity of erythroid spectrin.

Spectrin, the major protein of the erythrocyte membrane skeleton has canonically been thought to only serve a structural function. We have previously described a novel chaperone-like property of spectrin and also hypothesized that the chaperone activity and binding of a hydrophobic ligand, Prodan are localized in the self-association domain. Here we probe the location and molecular origin of the chaperone activity of multi-domain spectrin using a selection of individual recombinant spectrin domains, which we have characterized using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Aggregation assays using insulin, ADH, α- and ß-globin as well as enzyme refolding assays using alkaline phosphatase and α-glucosidase show that the chaperone activity is not only localized in the self-association domain but is a generalized property of spectrin domains. This is to our understanding, a unique feature in the case of modular multi-repeat proteins, possibly implicating that the large family of "spectrin-repeat" domain containing proteins may also have chaperone like property. Substrate selectivity of chaperone activity as evidenced by the preferential protection of α- over ß-globin chains is seen; which has implications in hemoglobin diseases. Moreover, enzyme-refolding assays also indicate alternate modes of chaperone action. We propose that the molecular origin of chaperone activity resides in the surface exposed hydrophobic patches of the spectrin domains as shown by ANS (1-anilinonaphthalene-8-sulfonic acid) and Prodan (6-propionyl-2[dimethylamino]-naphthalene) binding. We also show that Prodan does indeed have a unique binding site on spectrin located at the self-association domain.