Citations

Abstract Histone H3 lysine 9 methylation (H3K9me), which is written by the Euchromatic Histone Lysine Methyltransferases EHMT1 and EHMT2 and read by the heterochromatin protein 1 (HP1) chromobox (CBX) protein family, is dysregulated in many types of cancers. Approaches to inhibit regulators of this pathway are currently being evaluated for therapeutic purposes. Thus, knowledge of the complexes supporting the function of these writers and readers during the process of cell proliferation is critical for our understanding of their role in carcinogenesis. Here, we immunopurified each of these proteins and used mass spectrometry to define their associated non-histone proteins, individually and at two different phases of the cell cycle, namely G1/S and G2/M. Our findings identify novel binding proteins for these writers and readers, as well as corroborate known interactors, to show the formation of distinct protein complex networks in a cell cycle phase-specific manner. Furthermore, there is an organizational switch between cell cycle phases for interactions among specific writer–reader pairs. Through a multi-tiered bioinformatics-based approach, we reveal that many interacting proteins exhibit histone mimicry, based on an H3K9-like linear motif. Gene ontology analyses, pathway enrichment, and network reconstruction inferred that these comprehensive EHMT and CBX-associated interacting protein networks participate in various functions, including transcription, DNA repair, splicing, and membrane disassembly. Combined, our data reveals novel complexes that provide insight into key functions of cell cycle-associated epigenomic processes that are highly relevant for better understanding these chromatin-modifying proteins during cell cycle and carcinogenesis.

Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by digestion of the dissected glomerular proteins by trypsin, and subsequently mass spectrometry to identify and semiquantitate the glomerular proteins. LCM/MS has played a crucial role in the identification of novel types of amyloidosis, biomarker discovery in fibrillary GN, and more recently discovery of novel target antigens in membranous nephropathy (MN). In addition, LCM/MS has also confirmed the role for complement proteins in glomerular diseases, including C3 glomerulopathy. LCM/MS is now widely used as a clinical test and considered the gold standard for diagnosis and typing amyloidosis. For the remaining glomerular diseases, LCM/MS has remained a research tool. In this review, we discuss the usefulness of LCM/MS in other glomerular diseases, particularly MN, deposition diseases, and diseases of complement pathways, and advocate more routine use of LCM/MS at the present time in at least certain diseases, such as MN, for target antigen detection. We also discuss the limitations of LCM/MS, particularly the difficulties faced from moving from a research-based technique to a clinical test. Nonetheless, the role of LCM/MS in glomerular diseases is expanding. Currently, LCM/MS may be used to identify the etiology in certain glomerular diseases, but in the future, LCM/MS can play a valuable role in determining pathways of complement activation, inflammation, and fibrosis.

We present optimized settings for ramp times and ion mobility range in trapped ion mobility spectrometry experiments involving single cell analysis.

Significance Statement Syphilis is a common worldwide sexually transmitted infection. Proteinuria may occur in patients with syphilis. Membranous nephropathy (MN) is the most common cause of proteinuria in syphilis. The target antigen of MN in syphilis is unknown. This study shows that MN in syphilis is associated with a novel target antigen called neuron-derived neurotrophic factor (NDNF). NDNF-associated MN has distinctive clinical and pathologic manifestations and NDNF appears to be the target antigen in syphilis-associated MN. Background Syphilis is a common sexually transmitted infection. Membranous nephropathy (MN) is a common cause of proteinuria in syphilis. The target antigen is not known in most cases of syphilis-associated MN. Methods We performed laser microdissection of glomeruli and mass spectrometry (MS/MS) in 250 cases (discovery cohort) of phospholipase A2 receptor–negative MN to identify novel target antigens. This was followed by immunohistochemistry/confocal microscopy to localize the target antigen along the glomerular basement membrane (GBM). Western blot analyses using IgG eluted from frozen biopsy tissue were performed to detect binding to target antigen. Results MS/MS studies of the discovery cohort revealed high total spectral counts of a novel protein, neuron-derived neurotrophic factor (NDNF), in three patients: one each with syphilis and hepatitis B, HIV (syphilis status not known), and lung tumor. Next, MS/MS studies of five cases of syphilis-MN (validation cohort) confirmed high total spectral counts of NDNF (average 45±20.4) in all (100%) cases. MS/MS of 14 cases of hepatitis B were negative for NDNF. All eight cases of NDNF-associated MN were negative for known MN antigens. Electron microscopy showed stage I MN in all cases, with superficial and hump-like deposits without GBM reaction. IgG1 was the dominant IgG subtype on MS/MS and immunofluorescence microscopy. Immunohistochemistry/confocal microscopy showed granular staining and colocalization of NDNF and IgG along GBM. Western blot analyses using eluate IgG of NDNF-MN showed binding to both nonreduced and reduced NDNF, while IgG eluate from phospholipase A2 receptor-MN showed no binding. Conclusion NDNF is a novel antigenic target in syphilis-associated MN.

Abstract The existence of a murine homologue of the major basic protein (MBP) found in human eosinophil granules was initially hypothesized from structural similarities at the electron microscopic level. The results presented in this study have extended these observations by describing the identification/purification of a mouse MBP (mMBP) and the cloning of the gene encoding this eosinophil granule protein. Using protein purification methodologies with extravascular eosinophils, an mMBP homologue has been identified on the basis of strong (64%) N-terminal sequence homology with the mature human MBP (hMBP). Since hMBP results from a proteolytic cleavage of a precursor molecule, this sequence conservation suggests that the mouse granule protein is processed by a similar mechanism. The gene encoding mMBP was isolated using a hMBP cDNA clone as a heterologous probe in low criteria screens of mouse genomic and cDNA libraries. The genomic structure and nucleotide sequence of the mMBP exons are well conserved with the human gene, although homology alignments of the encoded proteins show that extensive sequence conservation occurs only in the mature portion of the MBP molecules. Expression data demonstrate that this gene is transcriptionally active in tissues containing eosinophil progenitor cells, such as femoral bone marrow. Genomic Southern blots using the mMBP gene at reduced stringency reveal the potential existence of a second, more divergent MBP-like sequence in the mouse. This suggests that, as with guinea pigs, the mouse genome may also encode the eosinophil major basic protein from more than one gene.