Nuclear Membrane Proteins with Potential Disease Links Found by Subtractive Proteomics

Schirmer, Eric C.; Florens, Laurence; Guan, Tinglu; Yates, John R.; Gerace, Larry

doi:10.1126/science.1088176

Cited by 607 publications

(610 citation statements)

References 21 publications

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“…A growing number of transmembrane proteins of the INM has been described, and several of these proteins interact with chromatin and/or lamins [2,3]. These interaction might be involved in targeting membranes to chromatin during the postmitotic reassembly of the NE [4][5][6].…”

Section: Introductionmentioning

confidence: 99%

The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology

et al. 2006

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The inner nuclear membrane (INM) of eukaryotic cells is characterized by a unique set of transmembrane proteins which interact with chromatin and/or the nuclear lamina. The number of identified INM proteins is steadily increasing, mainly as a result of proteomic and computational approaches. However, despite a link between mutation of several of these proteins and disease, the function of most transmembrane proteins of the INM remains unknown and depletion of many of these proteins from a variety of systems did not produce an obvious phenotype in the affected cells. Here, we report that depletion of the conserved INM protein Lem2 from human cell lines leads to abnormally shaped nuclei and severely reduces cell survival. We suggest that interactions of Lem2 with lamins or chromatin are critical for maintaining the integrity of the nuclear envelope.

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Section: Introductionmentioning

confidence: 99%

The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology

et al. 2006

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“…In an effort to differentiate between these possibilities, we first extracted NEs with the nonionic detergent Triton X-100. This treatment releases most NE membrane proteins with the exception of those that are tightly associated with the pelletable fraction containing NPCs attached to the lamina (Schirmer et al, 2003). To compare the extractability of various nups from the lamina, the pellet fraction was then extracted with increasing concentrations of NaCl.…”

Section: Nup53 Is Tightly Associated With the Nuclear Membrane And Lamentioning

confidence: 99%

Vertebrate Nup53 Interacts with the Nuclear Lamina and Is Required for the Assembly of a Nup93-containing Complex

Hawryluk-Gara

Shibuya

Wozniak

2005

MBoC

125

149

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The nuclear pore complex (NPC) is an evolutionarily conserved structure that mediates exchange of macromolecules across the nuclear envelope (NE). It is comprised of ϳ30 proteins termed nucleoporins that are each present in multiple copies. We have investigated the function of the human nucleoporin Nup53, the ortholog of Saccharomyces cerevisiae Nup53p. Both cell fractionation and in vitro binding data suggest that Nup53 is tightly associated with the NE membrane and the lamina where it interacts with lamin B. We have also shown that Nup53 is capable of physically interacting with a group of nucleoporins including Nup93, Nup155, and Nup205. Consistent with this observation, depletion of Nup53 using small interfering RNAs causes a decrease in the cellular levels of these nucleoporins as well as the spindle checkpoint protein Mad1, likely due to destabilization of Nup53-containing complexes. The cellular depletion of this group of nucleoporins, induced by depleting either Nup53 or Nup93, severely alters nuclear morphology producing phenotypes similar to that previously observed in cells depleted of lamin A and Mad1. On basis of these data, we propose a model in which Nup53 is positioned near the pore membrane and the lamina where it anchors an NPC subcomplex containing Nup93, Nup155, and Nup205. INTRODUCTIONThe nuclear envelope (NE) is a specialized membrane system that functions to separate the eukaryotic genome from the cytoplasm. The NE consists of an inner and an outer nuclear membrane. The former contains a distinct set of associated proteins, whereas the latter is structurally equivalent to the endoplasmic reticulum (reviewed in Mattaj, 2004). The nucleoplasmic face of the metazoan inner nuclear membrane is connected to a fibrous protein meshwork termed the nuclear lamina that forms a shell around the underlying chromatin mass. The lamina is composed of Aand B-type lamins, which are closely related to one another and to the intermediate filament-like family of proteins (reviewed in Burke, 2001). The nuclear lamina has been suggested to be involved in maintaining the structural integrity of the NE and influencing chromatin structure and function. The association of the lamina with transcriptionally inactive heterochromatin has led to the suggestion that it may play a role in regulating gene expression and some recent data support this idea (reviewed in Mattout-Drubezki and Gruenbaum, 2003).At numerous points along the NE the inner and outer nuclear membranes fuse to form pores ϳ100 nm in diameter. These pores are occupied by complex macromolecular structures termed nuclear pore complexes (NPCs). The NPCs are associated with euchromatin channels that extend into the interior of the nucleus, interrupting the continuity of the lamina and the heterochromatin. These complex structures have an estimated mass of ϳ60 million Daltons, but are composed of a relatively small number of proteins (ϳ30; Rout et al., 2000;Cronshaw et al., 2002) termed nucleoporins or nups. This is explained by the fact that all of these prot...

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“…Because each tandem mass spectrum represents an independent and verifiable piece of data, this approach to database searching has the ability to identify proteins in mixtures, enabling a rapid and comprehensive approach for the analysis of protein complexes and other complicated mixtures of proteins 6,[8][9][10][11][12] . New biology has been discovered based on fast and accurate protein identification [13][14][15][16][17][18] . As tandem mass spectral protein identification has proliferated, it has become increasingly important to understand the rationale of individual database search algorithms, their relative strengths and weaknesses, and the mathematics used to match sequence to spectrum.…”

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Large-scale database searching using tandem mass spectra: Looking up the answer in the back of the book

2004

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Database searching is an essential element of large-scale proteomics. Because these methods are widely used, it is important to understand the rationale of the algorithms. Most algorithms are based on concepts first developed in SEQUEST and PeptideSearch. Four basic approaches are used to determine a match between a spectrum and sequence: descriptive, interpretative, stochastic and probability-based matching. We review the basic concepts used by most search algorithms, the computational modeling of peptide identification and current challenges and limitations of this approach for protein identification.An unintended consequence of whole-genome sequencing has been the birth of large-scale proteomics. What drives proteomics is the ability to use mass spectrometry data of peptides as an 'address' or 'zip code' to locate proteins in sequence databases. Two mass spectrometry methods are used to identify proteins by database search methods. The first method uses a molecular weight fingerprint measured from a protein digested with a site-specific protease [1][2][3][4][5] . A second method uses tandem mass spectra derived from individual peptides of a digested protein 6,7 (Fig. 1). Because each tandem mass spectrum represents an independent and verifiable piece of data, this approach to database searching has the ability to identify proteins in mixtures, enabling a rapid and comprehensive approach for the analysis of protein complexes and other complicated mixtures of proteins 6,[8][9][10][11][12] . New biology has been discovered based on fast and accurate protein identification [13][14][15][16][17][18] . As tandem mass spectral protein identification has proliferated, it has become increasingly important to understand the rationale of individual database search algorithms, their relative strengths and weaknesses, and the mathematics used to match sequence to spectrum.In this review we discuss the prevailing fragmentation models, spectral preprocessing, methods to match tandem mass spectra to sequences and several approaches to matching tandem mass spectra of peptides whose exact sequences may not be present in the database. Space limitations restrict a detailed description of all algorithms in this rapidly expanding field. Also, some algorithms are proprietary, and thus, details on how they work are unknown. This review should supplement and update earlier reviews on database search algorithms [19][20][21][22][23][24] . Peptide fragmentation and data preprocessingIn tandem mass spectrometry (MS/MS), gas phase peptide ions undergo collision-induced dissociation (CID) with molecules of an inert gas such as helium or argon 25 . Other methods of dissociation have been developed, such as electron capture dissociation (ECD), surface induced dissociation (SID) and electron transfer dissociation (ETD), but gas-phase CID is the most widely used in commercial tandem mass spectrometers. The dissociation pathways are strongly dependent on the collision energy, but the vast majority of instruments use low-energy CID (<100 eV) 26 ....

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Nuclear Membrane Proteins with Potential Disease Links Found by Subtractive Proteomics

Cited by 607 publications

References 21 publications

The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology

The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology

Vertebrate Nup53 Interacts with the Nuclear Lamina and Is Required for the Assembly of a Nup93-containing Complex

Large-scale database searching using tandem mass spectra: Looking up the answer in the back of the book

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