Specific Binding Partners Research Articles

Immunostaining and Protein Pull-Down of Methyl-5-Cytosine-Marked RNA:DNA Hybrids.

RNA:DNA hybrids not only function in various physiological cellular processes but also represent a threat to genome integrity. The methyl-5-cytosine (m5C) marked RNA:DNA hybrids containing the m5C modified RNA strand add another layer of regulation to the R-loop structure. Here we describe the detection of m5C modifications in the context of RNA:DNA hybrids using immunostaining, and identification of specific binding partners of m5C marked RNA:DNA hybrids using pull-down method.

Precision medicine in aggressive thyroid cancer: Moving beyond multitargeted tyrosine kinase inhibitors

Precision medicine in aggressive thyroid cancer: Moving beyond multitargeted tyrosine kinase inhibitors

Ankyrin-repeat and SOCS box-containing protein 9 (ASB9) regulates ovarian granulosa cells function and MAPK signaling.

Ankyrin-repeat and SOCS box-containing proteins (ASB) interact with the elongin B-C adapter via their SOCS box domain and with the cullin and ring box proteins to form E3 ubiquitin ligase complexes within the protein ubiquitination pathway. ASB9 in particular is a differentially expressed gene in ovulatory follicles (OFs) induced by the luteinizing hormone (LH) surge or hCG injection in ovarian granulosa cells (GC) while downregulated in growing dominant follicles. Although ASB9 has been involved in biological processes such as protein modification, the signaling network associated with ASB9 in GC is yet to be fully defined. We previously identified and reported ASB9 interactions and binding partners in GC including PAR1, TAOK1, and TNFAIP6/TSG6. Here, we further investigate ASB9 effects on target binding partners regulation and signaling in GC. CRISPR/Cas9-induced inhibition of ASB9 revealed that ASB9 regulates PAR1, TAOK1, TNFAIP6 as well as genes associated with proliferation and cell cycle progression such as PCNA, CCND2, and CCNE2 while CCNA2 was not affected. Inhibition of ASB9 was also associated with increased GC number and decreased caspase3/7 activity, CASP3 expression, and BAX/BCL2 ratio. Furthermore, ASB9 induction in OF in vivo 24 h post-hCG is concomitant with a significant decrease in phosphorylation levels of MAPK3/1 while pMAPK3/1 levels increased following ASB9 inhibition in GC in vitro. Together, these results provide strong evidence for ASB9 as a regulator of GC activity and function by modulating MAPK signaling likely through specific binding partners such as PAR1, therefore controlling GC proliferation and contributing to GC differentiation into luteal cells.

Gain-of-Function of ASXL1 Truncating Protein in the Pathogenesis of Myeloid Malignancies

Introduction: Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We and others have reported that loss of ASXL1 impairs hematopoietic stem/progenitor cell functions (HSC/HPCs) and leads to myeloid dysplastic syndrome (MDS) in mice. It remains controversial whether truncating mutations in ASXL1 result in gain- or loss-of-function, or whether they confer dominant-negative activity in vivo . Answering this question is critical for determining how to target these mutations therapeutically.Methods: We generated a transgenic mouse model using HS321/45-vav vector that expresses the Asxl1 Y588X cDNA under the control of the Vav1 promoter. The transgene contains the entire coding region of mouse Asxl1 (full-length) with a stop codon mutation at the Y588 site (Asxl1 Y588X), leading to a production of truncating protein ASXL1aa1-587 in the hematopoietic system only. Utilizing the transgenic model, we performed a serial hematopoietic analyses, including peripheral blood (PB) counts, histological characterization on blood smear, tissue sections of femur and spleen, and flow cytometric analysis. Bone marrow(BM) transplantation was performed to identify the effects of truncating ASXL1aa1-587 on HSC/HPCs functions. RNA-seq and ATAC-seq analyses were performed to survey the differentially expressed genes in HSC/HPCs of WT and Asxl1 Y588X Tg mice. Liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation (IP) experiments were performed to identify the specific protein binding partner of ASXL1aa1-587. An epigenetic drug screening was used to identify specific targets to block the gain-of-function protein, ASXL1aa1-587, in the pathogenesis of myeloid malignancies in vivo.Results: The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patientswith ASXL1 mutations. Competitive transplantation of WT and Asxl1Y588XTg BM cells revealed that expression of A SXL 1 aa587 was associated with a higher percentage of CD45.2+ cells in the PB of the recipients, indicating that A SXL11-587aa expression increases HSC self-renewal capacity in vitro and in vivo. ATAC- and RNA-Seq analyses revealed that the ASXL11-587aa truncating protein expression results in more open chromatin in cKit+ cells compared to wildtype cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. LC-MS) and co-IP experiments showed that ASXL11-587aa acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hyper-sensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors.Conclusion: This study demonstrates that ASXL11-587aa plays a gain-of-function role in promoting myeloid malignancies and that BET bromodomain inhibitors significantly reduced the colony forming capacity of ASXL1Y588XTgHSPCs. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Ankyrin‐repeat and SOCS‐box Protein 9 (ASB9) regulate Ovarian Granulosa Cells function and MAPK signaling

Ankyrin-repeat and SOCS-box Protein 9 (ASB9) was previously identified as a differentially expressed gene in ovulatory follicles post-luteinizing hormone (LH) surge or hCG injection in ovarian granulosa cells (GC). ASB9 interactions in GC were also identified and included PAR1, TNFAIP6/TSG6, and TAOK1. However, ASB9 mechanisms of action in GC is still undefined. We investigated the role of ASB9 in GC through specific binding partners, its effects on MAPK signaling and on GC proliferation. An in vivo modelof GC from follicles at different developmental stages, small follicles (SF), dominant follicles (DF), and ovulatory follicles (OF) and an in vitro model of cultured GC along with the CRISPR/Cas9 approach to inhibit ASB9 were used. Inhibition of ASB9 in GC resulted in significant increase in PAR1, PCNA, CCND2, and CCNE2 and significant decrease in TAOK1, TNFAIP6, and CASP3 expression. From in vivo samples, PAR1 was differentially expressed in DF as compared to OF while TSG6 and TAOK1 were induced in OF. Further analyses showed an increase in GC number and a decrease in apoptosis and caspase3/7 activity following ASB9 inhibition. Western blot analyses demonstrated that ASB9 induction in OF by hCG from 12h post-hCG through 24h was concomitant with a significant decrease in MAPK3/1 phosphorylation levels while pMAPK3/1 increased following ASB9 inhibition. These results provide strong evidence that ASB9 is a regulator of GC activity and function by modulating MAPK signaling pathway likely through specific binding partners such as PAR1, therefore controlling GC proliferation and contributing to GC differentiation into luteal cells.

A study on the secondary structure of the metalloregulatory protein CueR: effect of pH, metal ions and DNA.

The response of CueR towards environmental changes in solution was investigated. CueR is a bacterial metal ion selective transcriptional metalloregulator protein, which controls the concentration of copper ions in the cell. Although several articles have been devoted to the discussion of the structural and functional features of this protein, CueR has not previously been extensively characterized in solution. Here, we studied the effect of change in pH, temperature, and the presence of specific or non-specific binding partners on the secondary structure of CueR with circular dichroism (CD) spectroscopy. A rather peculiar reversible pH-dependent secondary structure transformation was observed, elucidated and supplemented with pKa estimation by PROPKA and CpHMD simulations suggesting an important role of His(76) and His(94) in this process. CD experiments revealed that the presence of DNA prevents this structural switch, suggesting that DNA locks CueR in the α-helical-rich form. In contrast to the non-cognate metal ions HgII, CdII and ZnII, the presence of the cognate AgI ion affects the secondary structure of CueR, most probably by stabilizing the metal ion and DNA-binding domains of the protein.

PML-II recruits ataxin-3 to PML-NBs and inhibits its deubiquitinating activity

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic and multiprotein complexes implicated in a variety of important biochemical events. Due to alternative mRNA splicing, PML has at least six nuclear isoforms that share a common N-terminus but differ in their C-terminal regions. However, the unique role of each PML isoform is not clear. Here, we report the characterization of the deubiquitinase ataxin-3 as a specific binding partner of PML isoform II (PML-II). Ataxin-3 was identified as a potential binding protein of PML-II in a yeast-hybrid screen employing the unique C-terminal region of PML-II as bait. Ataxin-3 only binds to the C-terminal region of PML-II and not that of other PML isoforms. The interaction between ataxin-3 and PML-II was confirmed by co-immunoprecipition assays, and immunofluorescent microscopy revealed that PML-II and ataxin-3 were co-localized in PML-NBs. In addition, PML-II not only interacts with ataxin-3 with a normal range of poly-Q repeats (13Q), but also with a pathological form of ataxin-3 with extended poly-Q repeats (79Q). Importantly, the deubiquitinase activity of ataxin-3 was inhibited by PML-II. Our results suggest that PML-II may be a negative regulator of ataxin-3.

Multifunctionality and intrinsic disorder of royal jelly proteome.

Royal Jelly (RJ) is a gelatinous white-yellowish fluid, possessing a sour taste and a slight phenolic smell that is secreted by the hypopharyngeal and mandibular salivary glands of the nurse honeybees, and is used in nutrition of larvae and adult queens. Similar to other substances associated with the activities of honeybees, RJ not only contains nutritive components, such as carbohydrates, proteins, peptides, lipids, vitamins, and mineral salts,but also represents a natural ingredient with cosmetic and health-promoting properties. RJ is characterized by remarkable multifunctionality, possessing numerous biological activities. Although this multifunctionality of RJ can be considered as a consequence of its complex nature, many proteins and peptides in RJ are polyfunctional entities themselves. In this article, we show that RJ proteins contain different levels of intrinsic disorder, have sites of post-translational modifications, can be found in multiple isoforms, and many of them possess disorder-based binding sites, suggesting that the conformational ensembles of the RJ proteins might undergo change as a result of their interaction with specific binding partners. All these observations suggest that the multifunctionality of proteins and peptides from RJ is determined by their structural heterogeneity and polymorphism, and serve as an illustration of the protein structure-function continuum concept.

Characterization of SCF Ligase Inhibition by Tumor Suppressor HINT1

Skp, Cullin, F-box containing (SCF) ubiquitin E3 ligases are part of the largest family of E3 ligases. They ubiquitylate key proteins in many biological pathways. in recent years, in vivo studies have shown that the protein Histidine triad nucleotide-binding protein 1 (HINT1) inhibits the ubiquitylation activity of various SCF ligases that regulate the NF-κB signaling pathway and cell cycle progression by promoting the degradation of substratesIκBα and p27Kip1, respectively. However, the mechanism of interaction between HINT1 and SCF ligases and how HINT1 causes their inhibition is not understood. Many questions remain unanswered such as the specific binding partner of HINT1 within the SCF complex and how HINT1 is able to inhibit the activity of unique SCF ligases. in total, the ∼173 kDa SCF ligase complex consists of a scaffolding protein, Cullin1 (90 kDa), that utilizes Rbx1 (12 kDa) to recruit E2 ubiquitin conjugating enzymes (e.g. UBE2D2) and substrates through the adaptor and substrate receptor proteins, Skp1 (17 kDa) and β-TrCP1 (54 kDa) respectively, to promote substrate ubiquitylation. Through co-elution in gel-filtration chromatography, I have assembled the SCF E3 ligase that ubiquitylates IκBα and have demonstrated that HINT1 (28 kDa) binds to this ligase. I have performed biochemical experiments to measure ubiquitylation combined with HDX-MS and other biophysical experiments to characterize the binding affinity and the specific binding interface of HINT1 on the SCF E3 ligase. The results shed light on the molecular mechanism of inhibition of SCF E3 ligases by HINT1.

Molecular simulations of IDPs: From ensemble generation to IDP interactions leading to disorder-to-order transitions.

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure but do exhibit some dynamical and structural ordering. The structural plasticity of IDPs indicates that entropy-driven motions are crucial for their function. Many IDPs undergo function-related disorder-to-order transitions upon by their interaction with specific binding partners. Approaches that are based on both experimental and theoretical tools enable the biophysical characterization of IDPs. Molecular simulations provide insights into IDP structural ensembles and disorder-to-order transition mechanisms. However, such studies depend strongly on the chosen force field parameters and simulation techniques. In this chapter, we provide an overview of IDP characteristics, review all-atom force fields recently developed for IDPs, and present molecular dynamics-based simulation methods that allow IDP ensemble generation as well as the characterization of disorder-to-order transitions. In particular, we introduce metadynamics, replica exchange molecular dynamics simulations, and also kinetic models resulting from Markov State modeling, and provide various examples for the successful application of these simulation methods to IDPs.

Influenza A virus protein PA-X suppresses host Ankrd17-mediated immune responses.

Influenza A virus (IAV) PA-X is a critical ribonuclease protein involved in host cell shutoff but its role in modulating the host immune response to IAV infection remains to be addressed. In this study, host cellular proteins that directly interact with PA-X were screened to investigate the biological function of PA-X in the pathogenesis of IAV infection. The protein ankyrin repeat domain 17 (Ankrd17), a positive regulator of inflammatory responses via the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, was identified as a specific PA-X binding partner that preferred PA-X to the PA protein. The N-terminal ankyrin repeats of Ankrd17 are the key domain for the interaction with PA-X rather than PA, which is required for the function of Ankrd17 in elevating the host immune response. Using Ankrd17 knockout and overexpression, we confirmed that PA-X significantly affected the Ankrd17-mediated response to infection in host cells. Our data therefore reveal a novel function for PA-X in the regulation of innate immune pathways via the interaction between PA-X and Ankrd17.

Immunoglobulin-fold containing bacterial adhesins: molecular and structural perspectives in host tissue colonization and infection.

Immunoglobulin (Ig) domains are one of the most widespread protein domains encoded by the human genome and are present in a large array of proteins with diverse biological functions. These Ig domains possess a central structure, the immunoglobulin-fold, which is a sandwich of two β sheets, each made up of anti-parallel β strands, surrounding a central hydrophobic core. Apart from humans, proteins containing Ig-like domains are also distributed in a vast selection of organisms including vertebrates, invertebrates, plants, viruses and bacteria where they execute a wide array of discrete cellular functions. In this review, we have described the key structural deviations of bacterial Ig-folds when compared to the classical eukaryotic Ig-fold. Further, we have comprehensively grouped all the Ig-domain containing adhesins present in both Gram-negative and Gram-positive bacteria. Additionally, we describe the role of these particular adhesins in host tissue attachment, colonization and subsequent infection by both pathogenic and non-pathogenic Escherichia coli as well as other bacterial species. The structural properties of these Ig-domain containing adhesins, along with their interactions with specific Ig-like and non Ig-like binding partners present on the host cell surface have been discussed in detail.

Proteomics studies to investigate alterations in the tau interactome under conditions of elevated cellular O‐GlcNAcylation

AbstractBackgroundNeurofibrillary tangles consist of tau protein aggregates and represent a hallmark pathology of Alzheimer’s disease. Tau aggregation, specifically the formation of early aggregates, is believed to be neurotoxic. O‐linked glycosylation of proteins with β‐linked N‐acetylglucosamine (O‐GlcNAcylation) is exclusively mediated by two enzymes in cells, O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). Importantly, increasing O‐GlcNAcylation attenuates tau aggregation in cell‐free systems and cells and reduces tau pathology in various animal models, but the underlying molecular mechanism is still unclear. We aimed to determine how protein‐protein interactions of tau are affected by altered O‐GlcNAcylation levels in cells.Method(1) Immunoprecipitation of tau complexes and nano LC‐MS/MS analysis (IP‐MS) were performed to detect changes in tau binding proteins in HEK293T cells, upon elevated O‐GlcNAcylation by OGT overexpression and OGA inhibition. (2) Proximity labeling by TurboID, an engineered biotin ligase, fused to either wild‐type or O‐GlcNAcylation‐resistant mutant tau and nano LC‐MS/MS analysis were performed to assess changes in the proximity of tau to other proteins in iPSC‐derived human cortical neurons (iN), after increasing O‐GlcNAcylation by OGA inhibition.Result(1) We detected proteins that constitutively or differentially interact with tau from IP‐MS experiments in HEK293T cells. Microtubule and cytoskeletal proteins were consistently bound to tau regardless of changes in cellular O‐GlcNAcylation. RNA binding proteins and ribosomal proteins increased but cytosolic/exosome proteins and chaperone proteins decreased their interaction with tau upon elevated cellular O‐GlcNAcylation. (2) Proximity labeling of proteins by TurboID‐tau in iN successfully identified microtubule‐associated proteins and proteins that were previously reported to interact with tau in the brain. Interestingly, six proteins significantly changed their proximity to O‐GlcNAcylation‐resistant mutant tau, whereas increased O‐GlcNAcylation by OGA inhibition did not change the proximity labeling profile of wild‐type or mutant tau proteins.ConclusionThese results demonstrate that increasing cellular O‐GlcNAcylation does not significantly alter the general subcellular localization of tau, but affects its interaction with specific binding partners, some of which were previously implicated to play important a role in neurodegeneration. These data may provide novel mechanistic insights into how O‐GlcNAcylation impacts tau pathology.

CaMKIIα-driven, phosphatase-checked postsynaptic plasticity via phase separation.

Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca2+ oscillations. Despite decades of extensive research, new mechanisms underlying CaMKIIα's function in synapses are still being discovered. Here, we discover that Shank3 is a specific binding partner for autoinhibited CaMKIIα. We demonstrate that Shank3 and GluN2B, via combined actions of Ca2+ and phosphatases, reciprocally bind to CaMKIIα. Under basal condition, CaMKIIα is recruited to the Shank3 subcompartment of postsynaptic density (PSD) via phase separation. Rise of Ca2+ concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca2+ removal. Protein phosphatases control the Ca2+-dependent shuttling of CaMKIIα between the two PSD subcompartments and PSD condensate formation. Activation of CaMKIIα further enlarges the PSD assembly and induces structural LTP. Thus, Ca2+-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.

EBF1 Exhibits Crosstalk Regulation with ERα and ERβ in Some Hormone-Based Cancers

Estrogen-based cancers affect a substantial portion of the female population in the United States. While multiple studies have examined the effect of estrogen receptor alpha (ERα/ESR1) in cancer, the effects of ERβ/ESR2 are not as well understood in tumor tissues. Furthermore, there are few studies examining the role of specific binding partners of the estrogen receptors, such as early B-cell factor 1 (EBF1). EBF1 has been shown to have a role in B cell development and differentiation and is also known to bind to and crosstalk with both ERα and ERβ. However, to date those studies have only been performed in vitro in one breast cancer cell line. Therefore, it is imperative that we determine if there is a clinical role for that interaction. To that end, we utilized a bioinformatics approach to examine EBF1, ESR1, and ESR2 gene expression levels in patient tumor tissues from estrogen-based cancers of the breast, cervix, ovary, and uterus. Our results show a correlation between EBF1 and ESR1 or ESR2 expression, as well as some crosstalk regulation between EBF1 and ESR2, but the relationship between EBF1 and ESR1 is inconsistent across cancers. Furthermore, contrary to previous reports, we did not find a significant loss of EBF1 expression across all cancer types. Taken together, these results indicate that there is still much we do not understand about this interaction in clinical samples and provide an area for future studies.

Crystal structure of a 123 amino acids dimerization domain of Drosophila Caprin.

Cytoplasmic activation/proliferation-associated protein (Caprin) proteins assume diverse functions in many important biological processes, including synaptic plasticity, stress response, innate immune response, and cellular proliferation. The Caprin family members are characterized by the presence of a highly conserved homologous region (HR1) at the N-terminus and arginine-glycine-rich (RGG) boxes at the C-terminus. We had previously determined the crystal structures of human Caprin-1 and Caprin-2 fragments corresponding to the C-terminal 2/3 of HR1. Both fragments adopt homodimeric structures. Based on sequence conservation, we speculated that all Caprin proteins should have similar homodimeric structures. Here we report the crystal structure of a fragment (residues 187-309) of Drosophila melanogaster Caprin (dCaprin). The dCaprin fragment adopts an all α-helical fold which self-associates to form a homodimer. The overall dCaprin homodimeric structure is similar to the Caprin-1 and Caprin-2 homodimeric structures. Most of the amino acids residues mediating homodimerization in the three structures are conserved among all Caprin family members. These structural and sequence data suggest that homodimerization through a conserved dimerization domain is a common structural feature of the Caprin protein family. The dimeric structures may also be involved in interaction with Caprin partners. Dimer formation creates a V-shape concave surface that may serve as a protein binding groove. The concave surfaces in Caprin-1, Caprin-2, and dCaprin should have different and specific binding partners due to the large difference in electrostatic potentials. We propose the existence of a multi-functional domain in Caprin proteins, which not only mediate homodimerization but also involve in interaction with specific Caprin partners.

Parthenium hysterophorus mediated inflammation and hyper-responsiveness via NF-κB pathway in human A549 lung cancer cell line.

Being one of the notorious weed P. hysterophorus has invaded almost every part India and is the lead cause of skin allergies and severe dermatitis among farmers and rural population. It is an invasive obnoxious weed capable of surviving extreme environmental conditions and various parts of this plant are reported to cause severe contact allergies in humans due to the presence of high concentrations of toxic sesquiterpene lactones viz. parthenin. It can stimulate numerous cellular and immune responses that may translate into Oxidative stress, allergies, and inflammation. The effect of P. hysterophorus flower extract was evaluated on cell viability, oxidative stress and inflammation in A549 lung cancer cell line by spectrophotometric and reverse transcriptase-polymerase chain reaction methods. Schrodinger software based docking was performed for possible interactions studies. The A549 cells treated with P. hysterophorus flower extract favors increase in cell viability, reactive oxygen species generation. The mRNA expression of proinflammatory cytokines such as IFN-γ, TNF-α, and IL-1β was significantly increased whereas no change in IL-18 expression was observed. Significant increase in protein expression of NF-κB was observed, suggesting the role of NF-κB signalling in allergic responses. The docking studies demonstrated the potential interaction between Parthenin and NF-κB/IL-1β/IL-18 suggesting their activation leading to inflammation. The current study emphasize that P. hysterophorus mediates oxidative stress, and inflammatory process via alterations in expression of proinflammatory cytokines such as IL-1β, IFN-γ through NF-κB activation which was also confirmed in docking studies. Cellular and molecular mechanisms involved in pathogenesis of allergic/chronic inflammation and severe dermatitis need to be further investigated to identify specific binding partners responsible for severe inflammation which can provide some leads in developing effective targets against severe dermatitis and skin allergies.

Identification of the COMM-domain containing protein 1 as specific binding partner for the guanine-rich RNA sequence binding factor 1

BackgroundThe guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the hnRNP H/F family, which has been implicated in erythropoiesis, regulation of the redox homeostasis, embryonic brain development, mitochondrial function and cellular senescence. The molecular basis for GRSF1-RNA interaction has extensively been studied in the past but for the time being GRSF1 binding proteins have not been identified. MethodsTo search for GRSF1 binding proteins we first employed the yeast two-hybrid system and screened a cDNA library of human fetal brain for potential GRSF1 binding proteins. Subsequently, we explored the protein-protein-interaction of the recombiant proteins, carried out immunoprecipitation experiments to confirm the interaction of the native proteins in living cells and performed truncation studies to identify the protein-binding motif of GRSF1. ResultsUsing the yeast two-hybrid system we identified the COMM-domain containing protein 1 (COMMD1) as specific GRSF1 binding protein and in vitro truncation studies suggested that COMMD1 interacts with the alanine-rich domain of GRSF1. Co-immunoprecipitation strategies indicated that COMMD1-GRSF1 interaction was RNA independent and also occurred in living cells expressing the two native proteins. ConclusionIn mammalian cells the COMM-domain containing protein 1 (COMMD1) specifically interacts with the Ala-rich domain of GRSF1 in an RNA-independent manner. General significanceThis is the first report describing a specific GRSF1 binding protein.

Soft mechano-chemistry of molecular hubs in mitotic spindle: biomechanics and mechanical proofreading at microtubule ends

A microtubule (MT) is a long stiff tube-shaped filament formed by a hierarchical organization of a large number of tubulin protein molecules. These filaments constitute a major structural component of the scaffold of a multi-component macromolecular machine called mitotic spindle. The plus ends of the MTs are tethered to some specific binding partners by molecular tethers while those of some others are crosslinked by crosslinking molecules. Because of the non-covalent binding involved in the tethering and crosslinking, the attachments formed are intrinsically ‘soft’. These attachments are transient because these can get ruptured spontaneously by thermal fluctuations. By implementing in silico the standard protocols of in vitro molecular force spectroscopy, we compute the lifetimes of simple theoretical models of these attachments. The mean lifetime is essentially a mean first-passage time. The stability of cross-linked antiparallel MTs is shown to decrease monotonically with increasing tension, a characteristic of all ‘slip-bonds’. This is in sharp contrast to the nonmonotonic variation of the mean lifetime with tension, a mechanical fingerprint of ‘catch-bonds’, displayed by the MTs tethered to two distinct binding partners. We mention plausible functional implications of these observations in the context of mechanical proofreading.

Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα.

Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.