Structure and Function of Protein Arginine Methyltransferase PRMT7

PRMT7 is a member of the protein arginine methyltransferase (PRMT) family, which methylates a diverse set of substrates. Arginine methylation as a posttranslational modification regulates protein–protein and protein–nucleic acid interactions, and as such, has been implicated in various biological functions. PRMT7 is a unique, evolutionarily conserved PRMT family member that catalyzes the mono-methylation of arginine. The structural features, functional aspects, and compounds that inhibit PRMT7 are discussed here. Several studies have identified physiological substrates of PRMT7 and investigated the substrate methylation outcomes which link PRMT7 activity to the stress response and RNA biology. PRMT7-driven substrate methylation further leads to the biological outcomes of gene expression regulation, cell stemness, stress response, and cancer-associated phenotypes such as cell migration. Furthermore, organismal level phenotypes of PRMT7 deficiency have uncovered roles in muscle cell physiology, B cell biology, immunity, and brain function. This rapidly growing information on PRMT7 function indicates the critical nature of context-dependent functions of PRMT7 and necessitates further investigation of the PRMT7 interaction partners and factors that control PRMT7 expression and levels. Thus, PRMT7 is an important cellular regulator of arginine methylation in health and disease

A covalent homodimer probing early oligomers along amyloid aggregation

Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (beta2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing beta2m molecules was repeatedly observed, suggesting that such interface may be relevant for beta2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized beta2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt beta2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of beta2m aggregation. The present data provide new insight into beta2m early steps of amyloid aggregation

Wild type beta-2 microglobulin and DE loop mutants display a common fibrillar architecture

Beta-2 microglobulin (\u3b22m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining \u3b22m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity. However, it remains unclear whether the aggregates formed by wild type (wt) \u3b22m and by the DE loop variants are of the same kind, or whether the mutations open new aggregation pathways. In order to address this question, fibrillar samples of wt and mutated \u3b22m variants have been analysed by means of atomic force microscopy and infrared spectroscopy. The data here reported indicate that the DE loop mutants form aggregates with morphology and structural organisation very similar to the wt protein. Therefore, the main effect of \u3b22m DE loop mutations is proposed to stem from the different stabilities of the native fold. Considerations on the structural role of the DE loop in the free monomeric \u3b22m and as part of the Major Histocompatibility Complex are also presented

ATR/FTIR characterisation of DE loop mutants in the fibrillar state.

<p>A) The absorption spectra of the D59P fibrils were collected before and after incubation in D₂O for different times. Spectra are reported in the regions of Amide I, Amide II, and Amide II’ bands. Arrows point to the spectral changes at increased incubation time in D₂O. Absorption spectra are normalized at the Amide I maximum. B) Second derivatives of the absorption spectra of (A) in the Amide I region. The spectra collected after D₂O additions were normalized at the tyrosine band [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122449#pone.0122449.ref027" target="_blank">27</a>]. The peak positions of the main components are indicated. C) The absorption spectra of the W60V fibrils were collected before and after incubation in D₂O for different times and reported as in (A). D) Second derivatives of the absorption spectra of (C) in the Amide I region. E) Time course of the peak positions of the main intermolecular β-sheet component of wt, D59P, and W60V amyloid fibrils are reported after D₂O addition to the protein films. Error bars represent the standard deviation of at least three independent fibril preparations. The peak positions were taken from the second derivative spectra. F) The absorption spectra of W60G, wt, D59P, and W60V fibrils and that of W60G supernatant are reported in the Amide I region. The intermolecular β-sheet structure absorption band is marked. G) Second derivative spectra of the W60G supernatant collected before and after 23 hours from D₂O addition. The peak positions of the main components are indicated.</p

DE loop in monomeric β2m and in interaction within the MHC-I.

<p>(A) Ribbon representation of monomeric β2m (PDB code 2YXF). The DE loop residues are shown in yellow sticks. (B) Stereo view of the DE loop and Phe56 (yellow sticks) when interacting with the heavy chain in the MHC-I (electrostatic surface and green sticks). Trp60 is establishing a H-bond with Asp122 from the heavy chain (PDB code 4L29).</p

Investigating huntingtin DNA binding – plasmid EMSA with full-length HTT Q23/Q46

<p>Huntingtin structure-function open lab notebook.</p> <p> </p> <p>NB: plasmid concentration should read 0.5 mg/mL not 0.5 mg/uL.</p

Amyloid fibril formation of wt β2m, D59P, W60G and W60V β2m variants monitored by ThT fluorescence (given in arbitrary units).

<p>Amyloid fibril formation of wt β2m, D59P, W60G and W60V β2m variants monitored by ThT fluorescence (given in arbitrary units).</p

AFM characterisation of wt β2m and DE loop mutants aggregates incubated for one week.

<p>Tapping mode AFM images (height data) of mature fibrils of wt β2m and DE loop mutants obtained after one week incubation. Scan size 1.2 μm; the scale bars correspond to a Z range of: A and D) 55 nm; B) 70 nm; C) 65 nm. E-H) histograms of fibril height measured from fibril cross-sectional profiles in the topographic AFM images.</p

ATR/FTIR characterisation of DE loop mutants in the fibrillar state.

<p>A) The absorption spectra of the D59P fibrils were collected before and after incubation in D₂O for different times. Spectra are reported in the regions of Amide I, Amide II, and Amide II’ bands. Arrows point to the spectral changes at increased incubation time in D₂O. Absorption spectra are normalized at the Amide I maximum. B) Second derivatives of the absorption spectra of (A) in the Amide I region. The spectra collected after D₂O additions were normalized at the tyrosine band [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122449#pone.0122449.ref027" target="_blank">27</a>]. The peak positions of the main components are indicated. C) The absorption spectra of the W60V fibrils were collected before and after incubation in D₂O for different times and reported as in (A). D) Second derivatives of the absorption spectra of (C) in the Amide I region. E) Time course of the peak positions of the main intermolecular β-sheet component of wt, D59P, and W60V amyloid fibrils are reported after D₂O addition to the protein films. Error bars represent the standard deviation of at least three independent fibril preparations. The peak positions were taken from the second derivative spectra. F) The absorption spectra of W60G, wt, D59P, and W60V fibrils and that of W60G supernatant are reported in the Amide I region. The intermolecular β-sheet structure absorption band is marked. G) Second derivative spectra of the W60G supernatant collected before and after 23 hours from D₂O addition. The peak positions of the main components are indicated.</p