Crystal Structure of TDRD3 and Methyl-Arginine Binding Characterization of TDRD3, SMN and SPF30

SMN (Survival motor neuron protein) was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3) and SPF30 (Splicing factor 30 kDa) were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3

ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes*

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span

Enzymatic Activity Is Required for the in Vivo Functions of CARM1*

CARM1 is one of nine protein arginine methyltransferases that methylate arginine residues in proteins. CARM1 is recruited by many different transcription factors as a positive regulator. Gene targeting of CARM1 in mice has been performed, and knock-out mice, which are smaller than their wild-type littermates, die just after birth. It has been proposed that CARM1 has functions that are independent of its enzymatic activity. Indeed, CARM1 is found to interact with a number of proteins and may have a scaffolding function in this context. However, CARM1 methylates histone H3, PABP1, AIB1, and a number of splicing factors, which strongly suggests that its impact on transcription and splicing is primarily through its ability to modify these substrates. To unequivocally establish the importance of CARM1 enzymatic activity in vivo, we generated an enzyme-dead knock-in of this protein arginine methyltransferase. We determined that knock-in cells and mice have defects similar to those seen in their knock-out counterparts with respect to the time of embryo lethality, T cell development, adipocyte differentiation, and transcriptional coactivator activity. CARM1 requires its enzymatic activity for all of its known cellular functions. Thus, small molecule inhibitors of CARM1 will incapacitate all of the enzyme's cellular functions

Thrombospondin-1 Is a Transcriptional Repression Target of PRMT6*

Protein arginine methyltransferase 6 (PRMT6) is known to catalyze the generation of asymmetric dimethylarginine in polypeptides. Although the cellular role of PRMT6 is not well understood, it has been implicated in human immunodeficiency virus pathogenesis, DNA repair, and transcriptional regulation. PRMT6 is known to methylate histone H3 Arg-2 (H3R2), and this negatively regulates the lysine methylation of H3K4 resulting in gene repression. To identify in a nonbiased manner genes regulated by PRMT6 expression, we performed a microarray analysis on U2OS osteosarcoma cells transfected with control and PRMT6 small interfering RNAs. We identified thrombospondin-1 (TSP-1), a potent natural inhibitor of angiogenesis, as a transcriptional repression target of PRMT6. Moreover, we show that PRMT6-deficient U2OS cells exhibited cell migration defects that were rescued by blocking the secreted TSP-1 with a neutralizing peptide or blocking α-TSP-1 antibody. PRMT6 associates with the TSP-1 promoter and regulates the balance of methylation of H3R2 and H3K4, such that in PRMT6-deficient cells H3R2 was hypomethylated and H3K4 was trimethylated at the TSP-1 promoter. Using a TSP-1 promoter reporter gene, we further show that PRMT6 directly regulates the TSP-1 promoter activity. These findings show that TSP-1 is a transcriptional repression target of PRMT6 and suggest that neutralizing the activity of PRMT6 could inhibit tumor progression and therefore may be of cancer therapeutic significance

Sensing Tissue Damage by Myeloid C-Type Lectin Receptors

After both sterile and infectious insults, damage is inflicted on tissues leading to accidental or programmed cell death. In addition, events of programmed cell death also take place under homeostatic conditions, such as in embryo development or in the turnover of hematopoietic cells. Mammalian tissues are seeded with myeloid immune cells, which harbor a plethora of receptors that allow the detection of cell death, modulating immune responses. The myeloid C-type lectin receptors (CLRs) are one of the most prominent families of receptors involved in tailoring immunity after sensing dead cells. In this chapter, we will cover a diversity of signals arising from different forms of cell death and how they are recognized by myeloid CLRs. We will also explore how myeloid cells develop their sentinel function, exploring how some of these CLRs identify cell death and the type of responses triggered thereof. In particular, we will focus on DNGR-1 (CLEC9A), Mincle (CLEC4E), CLL-1 (CLEC12A), LOX-1 (OLR1), CD301 (CLEC10A) and DEC-205 (LY75) as paradigmatic death-sensing CLRs expressed by myeloid cells. The molecular processes triggered after cell death recognition by myeloid CLRs contribute to the regulation of immune responses in pathologies associated with tissue damage, such as infection, autoimmunity and cancer. A better understanding of these processes may help to improve the current approaches for therapeutic intervention.Carlos Del Fresno is supported by AECC Foundation (INVES192DELF). Francisco Javier Cueto is the recipient of a Ph.D. “La Caixa” fellowship (LCF/BQ/ES14/10320011). Work in the DS laboratory is funded by the CNIC; by the European Research Council (ERC-2016-Consolidator Grant 725091); by the European Commission (635122-PROCROP H2020); by Ministerio de Ciencia, Innovación e Universidades (MICINN), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-79040-R); by Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); by FIS-Instituto de Salud Carlos III, MICINN and FEDER (RD16/0015/0018-REEM); by Acteria Foundation; by Atresmedia (Constantes y Vitales prize) and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S