NSD2 is recruited through its PHD domain to oncogenic gene loci to drive multiple myeloma.

Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)- multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation.

Structure of human SMYD2 protein reveals the basis of p53 tumor suppressor methylation.

SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368-375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1-282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel ß7 and ß8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.

Efficient and clean charge derivatization of peptides for analysis by mass spectrometry.

Charge derivatization with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) has great potential in several aspects of proteomics, such as peptide de novo sequencing, PTM analysis, etc. However, the excess reagent and its side products greatly limited its scope of use. Here, we report an improved method to perform charge derivatization of peptides by TMPP-Ac-OSu without interference from the excess reagent and corresponding side products. Briefly, the protein was first separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or coagulated with the gel. The protein in-gel was then incubated with a high concentration of reagent, followed by extensive washing. Afterwards, the protein was in-gel digested with trypsin according to the routine protocol. The mainly resultant peptides were attached with one positive tag on the N-termini or Lys-epsilon-NH(2). The process has been successfully applied to 2-DE resolved protein spots. Compared to the native proteins, the derivatized counterparts have higher rates of PMF identification and more straightforward tandem mass spectra. This promising method should pave the way for the practical use of charge derivatization in proteomics.

Inflammation inhibitors were remarkably up-regulated in plasma of severe acute respiratory syndrome patients at progressive phase.

Severe acute respiratory syndrome (SARS) is a severe infectious disease that has affected many countries and regions since 2002. A novel member of the coronavirus, SARS-CoV, has been identified as the causative agent. However, the pathogenesis of SARS is still elusive. In this study, we used 2-D DIGE and MS to analyze the protein profiles of plasma from SARS patients, in the search for proteomic alterations associated with the disease progression, which could provide some clues to the pathogenesis. To enrich the low-abundance proteins in human plasma, two highly abundant proteins, albumin and IgG, were first removed. By comparing the plasma proteins of SARS patients with those of a normal control group, several proteins with a significant alteration were found. The up-regulated proteins were identified as alpha-1 acid glycoprotein, haptoglobin, alpha-1 anti-chymotrypsin and fetuin. The down-regulated proteins were apolipoprotein A-I, transferrin and transthyretin. Most of the proteins showed significant changes (up- or down-regulated) in the progressive phase of disease, and there was a trend back to normal level during the convalescent phase. Among these proteins, the alterations of fetuin and anti-chymotrypsin were further confirmed by Western blotting. Since all the up-regulated proteins identified above are well-known inflammation inhibitors, these results strongly suggest that the body starts inflammation inhibition to sustain the inflammatory response balance in the progression of SARS.

Enrichment and identification of cysteine-containing peptides from tryptic digests of performic oxidized proteins by strong cation exchange LC and MALDI-TOF/TOF MS.

The extreme complexity of sample and uninformative fragmentation of peptides in MS/MS experiments are two of several real challenges faced by proteomics. In this work, a strategy aimed at tackling these two problems is presented. Briefly, proteins were first oxidized by performic acid to cleave the disulfide bonds and simultaneously convert cysteine residue into its sulfonic form. Then the resultant sulfonic peptides were enriched by SCX chromatography, exploiting the negative solution charge of sulfonic group. The sulfonic peptide could be easily detected by MALDI-MS in negative mode and showed both enhanced fragmentation efficiency and a simplified spectrum in MALDI-MS/MS experiment in positive mode. The strength of the strategy was demonstrated by applying it to bovine serum albumin. Potential use of the strategy in proteomics was also discussed.

Identification of degradation products formed during performic oxidation of peptides and proteins by high-performance liquid chromatography with matrix-assisted laser desorption/ionization and tandem mass spectrometry.

Oxidation of proteins with performic acid is extensively used to cleave disulfide bonds. Due to its efficiency and many other advantages it deserves more attention especially in proteomics as a method for sample treatment. However, some unwanted degradations can occur during performic oxidation. In this work the degradation products during performic oxidation of two peptides and bovine serum albumin as model substrates were explored by coupling high-performance liquid chromatography (HPLC) to matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF/TOFMS). In addition to well-known modifications such as oxidation of tryptophan and oxidation and chlorination of tyrosine, novel degradation products including nonspecific cleavage after asparagine or tryptophan, formylation of lysine, and beta-elimination of cysteine, were observed. Although almost all of these modification/degradation products except oxidation products of tryptophan were formed at sub-stoichiometric levels, they can cause confusion as a result of the sensitivity of mass spectrometry in analysis of the oxidized samples, especially in proteomics research. The results presented here will facilitate the interpretation of analytical data for performate-oxidized samples, and help to select appropriate methods for each unique sample.

Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus.

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.