Comparative proteomic analyses of the nuclear envelope and pore complex suggests a wide range of heretofore unexpected functions

Since the discovery of several inherited diseases linked to the nuclear envelope the number of functions ascribed to this subcellular organelle has skyrocketed. However the molecular pathways underlying these functions are not clear in most cases, perhaps because of missing components. Several recent proteomic analyses of the nuclear envelope and nuclear pore complex proteomes have yielded not only enough missing components to potentially elucidate these pathways, but suggest an exponentially greater number of functions at the nuclear periphery than ever imagined. Many of these functions appear to derive from recapitulation of pathways utilized at the plasma membrane and from other membrane systems. Additionally, many proteins identified in the comparative nuclear envelope studies have sequence characteristics suggesting that they might also contribute to nuclear pore complex functions. In particular, the striking enrichment for proteins in the nuclear envelope fractions that carry phenylalanine-glycine (FG) repeats may be significant for the mechanism of nuclear transport. In retrospect, these findings are only surprising in context of the notion held for many years that the nuclear envelope was only a barrier protecting the genome. In fact, it is arguably the most complex membrane organelle in the cell.     

Diseases of the Nuclear Envelope

In the past decade, a wide range of fascinating monogenic diseases have been linked to mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins of the nuclear envelope. These diseases include dilated cardiomyopathy with variable muscular dystrophy, Dunnigan-type familial partial lipodystrophy, a Charcot-Marie-Tooth type 2 disease, mandibuloacral dysplasia, and Hutchinson-Gilford progeria syndrome. Several diseases are also caused by mutations in genes encoding B-type lamins and proteins that associate with the nuclear lamina. Studies of these so-called laminopathies or nuclear envelopathies, some of which phenocopy common human disorders, are providing clues about functions of the nuclear envelope and insights into disease pathogenesis and human aging.Mutations in LMNA encoding the A-type lamins cause a group of human disorders often collectively called laminopathies. The major A-type lamins, lamin A and lamin C, arise by alternative splicing of the LMNA pre-mRNA and are expressed in virtually all differentiated somatic cells. Although the A-type lamins are widely expressed, LMNA mutations are responsible for at least a dozen different clinically defined disorders with tissue-selective abnormalities. Mutations in genes encoding B-type lamins and lamin-associated proteins, most of which are similarly expressed in almost all somatic cells, also cause tissue-selective diseases.Research on the laminopathies has provided novel clues about nuclear envelope function. Recent studies have begun to shed light on how alterations in the nuclear envelope could explain disease pathogenesis. Along with basic research on nuclear structure, the nuclear lamins, and lamina-associated proteins, clinical research on the laminopathies will contribute to a complete understanding of the functions of the nuclear envelope in normal physiology and in human pathology.     

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, beta-sheet motifs and stretches of alpha-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.     

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, β-sheet motifs and stretches of α-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.     

Analysis of the Arabidopsis nuclear proteome and its response to cold stress

The nucleus is the subcellular organelle that contains nearly all the genetic information required for the regulated expression of cellular proteins. In this study, we comprehensively characterized the Arabidopsis nuclear proteome. Nuclear proteins were isolated and analyzed using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Approximately 500-700 spots were detected in reference 2D gels of nuclear proteins. Proteomic analyses led to the identification of 184 spots corresponding to 158 different proteins implicated in a variety of cellular functions. We additionally analyzed the changes in the nuclear proteome in response to cold stress. Of the 184 identified proteins, 54 were up- or downregulated with a greater than twofold change in response to cold treatment. Among these, six proteins were selected for further characterization. Northern analysis data revealed that gene expression of these proteins was also altered by cold stress. Following transient expression in BY-2 protoplasts, two proteins were detected in both the cytoplasm and the nucleus and four others were detected exclusively in the nucleus, which correlates well with the nuclear localization patterns of the proteomic data. Our study provides an initial insight into the Arabidopsis nuclear proteome and its response to cold stress.     

The secretome atlas of two mouse models of progeria

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by nuclear envelope alterations that lead to accelerated aging and premature death. Several studies have linked health and longevity to cell-extrinsic mechanisms, highlighting the relevance of circulating factors in the aging process as well as in age-related diseases. We performed a global plasma proteomic analysis in two preclinical progeroid models (Lmna^G609G/G609G and Zmpste24^−/− mice) using aptamer-based proteomic technology. Pathways related to the extracellular matrix, growth factor response and calcium ion binding were among the most enriched in the proteomic signature of progeroid samples compared to controls. Despite the global downregulation trend found in the plasma proteome of progeroid mice, several proteins associated with cardiovascular disease, the main cause of death in HGPS, were upregulated. We also developed a chronological age predictor using plasma proteome data from a cohort of healthy mice (aged 1–30 months), that reported an age acceleration when applied to progeroid mice, indicating that these mice exhibit an “old” plasma proteomic signature. Furthermore, when compared to naturally-aged mice, a great proportion of differentially expressed circulating proteins in progeroid mice were specific to premature aging, highlighting secretome-associated differences between physiological and accelerated aging. This is the first large-scale profiling of the plasma proteome in progeroid mice, which provides an extensive list of candidate circulating plasma proteins as potential biomarkers and/or therapeutic targets for further exploration and hypothesis generation in the context of both physiological and premature aging.     

Nuclear envelope disease and chromatin organization

The fifth U.K. meeting on nuclear envelope disease and chromatin brought together international experts from across the field of nuclear envelope biology to discuss the advancements in a class of tissue-specific degenerative diseases called the laminopathies. Clinically, these range from relatively mild fat-wasting disorders to the severe premature aging condition known as Hutchinson-Gilford progeria syndrome. Since the first association of the nuclear envelope with human inherited disease in 1994, there has been an exponential increase in an unexpected variety of functions associated with nuclear envelope proteins, ranging from mechanical support and nucleocytoskeletal connections to regulation of chromatin organization and gene expression. This Biochemical Society Focused Meeting reinforced the functional complexity of nuclear-associated diseases, revealed new avenues to be investigated and highlighted the signalling pathways suitable as therapeutic targets.     

The nuclear envelope in muscular dystrophy and cardiovascular diseases

Considerable interest has been focused on the nuclear envelope in recent years following the realization that several human diseases are linked to defects in genes encoding nuclear envelope specific proteins, most notably A-type lamins and emerin. These disorders, described as laminopathies or nuclear envelopathies, include both X-linked and autosomal dominant forms of Emery–Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction system defects, limb girdle muscular dystrophy 1B with atrioventricular conduction disturbances, and Dunnigan-type familial partial lipodystrophy. Certain of these diseases are associated with nuclear structural abnormalities that can be seen in a variety of cells and tissues. These observations clearly demonstrate that A-type lamins in particular play a central role, not only in the maintenance of nuclear envelope integrity but also in the large-scale organization of nuclear architecture. What is not obvious, however, is why defects in nuclear envelope proteins that are found in most adult cell types should give rise to pathologies associated predominantly with skeletal and cardiac muscle and adipocytes. The recognition of these various disorders now raises the novel possibility that the nuclear envelope may have functions that go beyond housekeeping and which impact upon cell-type specific nuclear processes.     

Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis

Background  

The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation.     

The laminopathies: nuclear structure meets disease

Most inherited diseases are associated with mutations in a specific gene. Sometimes, mutations in two or more different genes result in diseases with a similar phenotype. Rarely do different mutations in the same gene result in a multitude of seemingly different and unrelated diseases. In the past three years, different mutations in LMNA, the gene encoding the A-type lamins, have been shown to be associated with at least six different diseases. These diseases and at least two others caused by mutations in other proteins associated with the nuclear lamina are collectively called the laminopathies. How different tissue-specific diseases arise from unique mutations in the LMNA gene, encoding almost ubiquitously expressed nuclear proteins, are providing tantalizing insights into the structural organization of the nucleus, its relation to nuclear function in different tissues and the involvement of the nuclear envelope in the development of disease.     

Domain altering SNPs in the human proteome and their impact on signaling pathways

Single nucleotide polymorphisms (SNPs) constitute an important mode of genetic variations observed in the human genome. A small fraction of SNPs, about four thousand out of the ten million, has been associated with genetic disorders and complex diseases. The present study focuses on SNPs that fall on protein domains, 3D structures that facilitate connectivity of proteins in cell signaling and metabolic pathways. We scanned the human proteome using the PROSITE web tool and identified proteins with SNP containing domains. We showed that SNPs that fall on protein domains are highly statistically enriched among SNPs linked to hereditary disorders and complex diseases. Proteins whose domains are dramatically altered by the presence of an SNP are even more likely to be present among proteins linked to hereditary disorders. Proteins with domain-altering SNPs comprise highly connected nodes in cellular pathways such as the focal adhesion, the axon guidance pathway and the autoimmune disease pathways. Statistical enrichment of domain/motif signatures in interacting protein pairs indicates extensive loss of connectivity of cell signaling pathways due to domain-altering SNPs, potentially leading to hereditary disorders.     

Life at the edge: the nuclear envelope and human disease

A group of human diseases, known as 'laminopathies', are associated with defects in proteins of the nuclear envelope. Most laminopathy mutations have been mapped to the A-type lamin gene, which is expressed in most adult cell types. So, why should different mutations in a near-ubiquitously expressed gene be associated with various discrete tissue-restricted diseases? Attempts to resolve this paradox are uncovering new molecular interactions #151; both inside the nucleus and at its periphery -- which indicate that the nuclear envelope has functions that go beyond mere housekeeping.     

Nuclear envelope defects in muscular dystrophy

Muscular dystrophies are a heterogeneous group of disorders linked to defects in 20-30 different genes. Mutations in the genes encoding a pair of nuclear envelope proteins, emerin and lamin A/C, have been shown to cause the X-linked and autosomal forms respectively of Emery-Dreifuss muscular dystrophy. A third form of muscular dystrophy, limb girdle muscular dystrophy 1b, has also been linked to mutations in the lamin A/C gene. Given that these two genes are ubiquitously expressed, a major goal is to determine how they can be associated with tissue specific diseases. Recent results suggest that lamin A/C and emerin contribute to the maintenance of nuclear envelope structure and at the same time may modulate the expression patterns of certain mechanosensitive and stress induced genes. Both emerin and lamin A/C may play an important role in the response of cells to mechanical stress and in this way may help to maintain muscle cell integrity.     

Synaptopathies: diseases of the synaptome

The human synapse proteome is a highly complex collection of proteins that is disrupted by hundreds of gene mutations causing over 100 brain diseases. These synaptic diseases, or synaptopathies, cause major psychiatric, neurological and childhood developmental disorders through mendelian and complex genetic mechanisms. The human postsynaptic proteome and its core signaling complexes built by the assembly of receptors and enzymes around Membrane Associated Guanylate Kinase (MAGUK) scaffold proteins are a paradigm for systematic analysis of synaptic diseases. In humans, the MAGUK Associated Signaling Complexes are mutated in Autism, Schizophrenia, Intellectual Disability and many other diseases, and mice carrying orthologous mutations show relevant cognitive, social, motoric and other phenotypes. Diseases with similar phenotypes and symptom spectrums arise from disruption of complexes and interacting proteins within the synapse proteome. Classifying synaptic disease phenotypes with genetic and proteome data provides a new brain disease classification system based on molecular etiology and pathogenesis.     

Aging and nuclear organization: lamins and progeria

The discoveries of at least eight human diseases arising from mutations in LMNA, which encodes the nuclear A-type lamins, have revealed the nuclear envelope as an organelle associated with a variety of fundamental cellular processes. The most recently discovered diseases associated with LMNA mutations are the premature aging disorders Hutchinson-Gilford progeria syndrome (HGPS) and atypical Werner's syndrome. The phenotypes of both HGPS patients and a mouse model of progeria suggest diverse compromised tissue functions leading to defects reminiscent of aging. Aspects of the diseases associated with disrupted nuclear envelope/lamin functions may be explained by decreased cellular proliferation, loss of tissue repair capability and a decline in the ability to maintain a differentiated state.     

Subcellular fractionation enhances proteome coverage of pancreatic duct cells

ObjectivesSubcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage.MethodsWe used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared – via mass spectrometry-based analysis – the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates.ResultsWe identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle.ConclusionsSubcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.     

Protein profiling reveals energy metabolism and cytoskeletal protein alterations in LMNA mutation carriers

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.