Leveraging Parameter Dependencies in High-Field Asymmetric Waveform Ion-Mobility Spectrometry and Size Exclusion Chromatography for Proteome-wide Cross-Linking Mass Spectrometry.

Ion-mobility spectrometry shows great promise to tackle analytically challenging research questions by adding another separation dimension to liquid chromatography-mass spectrometry. The understanding of how analyte properties influence ion mobility has increased through recent studies, but no clear rationale for the design of customized experimental settings has emerged. Here, we leverage machine learning to deepen our understanding of field asymmetric waveform ion-mobility spectrometry for the analysis of cross-linked peptides. Knowing that predominantly m/z and then the size and charge state of an analyte influence the separation, we found ideal compensation voltages correlating with the size exclusion chromatography fraction number. The effect of this relationship on the analytical depth can be substantial as exploiting it allowed us to almost double unique residue pair detections in a proteome-wide cross-linking experiment. Other applications involving liquid- and gas-phase separation may also benefit from considering such parameter dependencies.

Structural disorder of monomeric α-synuclein persists in mammalian cells.

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-ß component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

The field of structural biology is increasingly focusing on studying proteins in situ, i.e., in their greater biological context. Cross-linking mass spectrometry (CLMS) is contributing to this effort, typically through the use of mass spectrometry (MS)-cleavable cross-linkers. Here, we apply the popular noncleavable cross-linker disuccinimidyl suberate (DSS) to human mitochondria and identify 5518 distance restraints between protein residues. Each distance restraint on proteins or their interactions provides structural information within mitochondria. Comparing these restraints to protein data bank (PDB)-deposited structures and comparative models reveals novel protein conformations. Our data suggest, among others, substrates and protein flexibility of mitochondrial heat shock proteins. Through this study, we bring forward two central points for the progression of CLMS towards large-scale in situ structural biology: First, clustered conflicts of cross-link data reveal in situ protein conformation states in contrast to error-rich individual conflicts. Second, noncleavable cross-linkers are compatible with proteome-wide studies.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2.

Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine ß-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time.

Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.

Nucleotide binding triggers a conformational change of the CBS module of the magnesium transporter CNNM2 from a twisted towards a flat structure.

Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2+ extruder at the renal distal convoluted tubule, or its regulator. In the present study, we explore structural features and ligand-binding capacities of the Bateman module of CNNM2 (residues 429-584), an intracellular domain structurally equivalent to the region involved in Mg2+ handling by the bacterial Mg2+ transporter MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the structural impact of the pathogenic mutation T568I located in this region. Our crystal structures reveal that nucleotides such as AMP, ADP or ATP bind at only one of the two cavities present in CNNM2429-584. Mg2+ favours ATP binding by alleviating the otherwise negative charge repulsion existing between acidic residues and the polyphosphate group of ATP. In crystals CNNM2429-584 forms parallel dimers, commonly referred to as CBS (cystathionine ß-synthase) modules. Interestingly, nucleotide binding triggers a conformational change in the CBS module from a twisted towards a flat disc-like structure that mostly affects the structural elements connecting the Bateman module with the transmembrane region. We furthermore show that the T568I mutation, which causes dominant hypomagnesaemia, mimics the structural effect induced by nucleotide binding. The results of the present study suggest that the T568I mutation exerts its pathogenic effect in humans by constraining the conformational equilibrium of the CBS module of CNNM2, which becomes 'locked' in its flat form.

Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis.

In the kidney, tight junction proteins contribute to segment specific selectivity and permeability of paracellular ion transport. In the thick ascending limb (TAL) of Henle's loop, chloride is reabsorbed transcellularly, whereas sodium reabsorption takes transcellular and paracellular routes. TAL salt transport maintains the concentrating ability of the kidney and generates a transepithelial voltage that drives the reabsorption of calcium and magnesium. Thus, functionality of TAL ion transport depends strongly on the properties of the paracellular pathway. To elucidate the role of the tight junction protein claudin-10 in TAL function, we generated mice with a deletion of Cldn10 in this segment. We show that claudin-10 determines paracellular sodium permeability, and that its loss leads to hypermagnesemia and nephrocalcinosis. In isolated perfused TAL tubules of claudin-10-deficient mice, paracellular permeability of sodium is decreased, and the relative permeability of calcium and magnesium is increased. Moreover, furosemide-inhibitable transepithelial voltage is increased, leading to a shift from paracellular sodium transport to paracellular hyperabsorption of calcium and magnesium. These data identify claudin-10 as a key factor in control of cation selectivity and transport in the TAL, and deficiency in this pathway as a cause of nephrocalcinosis.

Quantitative NMR analysis of Erk activity and inhibition by U0126 in a panel of patient-derived colorectal cancer cell lines.

We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia.

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.

Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation.

Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.

Membrane topology and intracellular processing of cyclin M2 (CNNM2).

Recently, mutations in the cyclin M2 (CNNM2) gene were identified to be causative for severe hypomagnesemia. In kidney, CNNM2 is a basolaterally expressed protein with predominant expression in the distal convoluted tubule. Transcellular magnesium (Mg(2+)) reabsorption in the distal convoluted tubule represents the final step before Mg(2+) is excreted into the urine, thus fine-tuning its final excretion via a tightly regulated mechanism. The present study aims to get insight in the structure of CNNM2 and to characterize its post-translational modifications. Here, membrane topology studies using intramolecular epitopes and immunocytochemistry showed that CNNM2 has an extracellular N terminus and an intracellular C terminus. This suggests that one of the predicted transmembrane regions might be re-entrant. By homology modeling, we demonstrated that the loss-of-function mutation as found in patients disturbs the potential ATP binding by the intracellular cystathionine ß-synthase domains. In addition, the cellular processing pathway of CNNM2 was exposed in detail. In the endoplasmic reticulum, the signal peptidase complex cleaves off a large N-terminal signal peptide of about 64 amino acids. Mutagenesis screening showed that CNNM2 is glycosylated at residue Asn-112, stabilizing CNNM2 on the plasma membrane. Interestingly, co-immunoprecipitation studies evidenced that CNNM2a forms heterodimers with the smaller isoform CNNM2b. These new findings on CNNM2 structure and processing may aid to elucidate the physiological role of CNNM2 in Mg(2+) reabsorption in the kidney.

Purification, crystallization and preliminary crystallographic analysis of the CBS-domain pair of cyclin M2 (CNNM2).

This work describes the purification and preliminary crystallographic analysis of the CBS-domain pair of the murine CNNM2 magnesium transporter (formerly known as ancient domain protein 2; ACDP2), which consists of a pair of cystathionine ß-synthase (CBS) motifs and has 100% sequence identity to its human homologue. CNNM proteins represent the least-studied members of the eight different types of magnesium transporters identified to date in mammals. In humans, the CNNM family is encoded by four genes: CNNM1-4. CNNM1 acts as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Interestingly, mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures and cardiac arrhythmias. This manuscript describes the preliminary crystallographic analysis of two different crystal habits of a truncated form of the protein containing its regulatory CBS-domain pair, which has been reported to host the pathological mutation T568I in humans. The crystals belonged to space groups P2(1)2(1)2 and I222 (or I2(1)2(1)2(1)) and diffracted X-rays to 2.0 and 3.6 Šresolution, respectively, using synchrotron radiation.

Targeted deletion of murine Cldn16 identifies extra- and intrarenal compensatory mechanisms of Ca2+ and Mg2+ wasting.

Claudin-16 (CLDN16) is critical for renal paracellular epithelial transport of Ca(2+) and Mg(2+) in the thick ascending loop of Henle. To gain novel insights into the role of CLDN16 in renal Ca(2+) and Mg(2+) homeostasis and the pathological mechanisms underlying a human disease associated with CLDN16 dysfunction [familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), OMIM 248250], we generated a mouse model of CLDN16 deficiency. Similar to patients, CLDN16-deficient mice displayed hypercalciuria and hypomagnesemia. Contrary to FHHNC patients, nephrocalcinosis was absent in our model, indicating the existence of compensatory pathways in ion handling in this model. In line with the renal loss of Ca(2+), compensatory mechanisms like parathyroid hormone and 1,25(OH)(2)D(3) were significantly elevated. Also, gene expression profiling revealed transcriptional upregulation of several Ca(2+) and Mg(2+) transport systems including Trpv5, Trpm6, and calbindin-D9k. Induced gene expression was also seen for the transcripts of two putative Mg(2+) transport proteins, Cnnm2 and Atp13a4. Moreover, urinary pH was significantly lower when compared with wild-type mice. Taken together, our findings demonstrate that loss of CLDN16 activity leads to specific alterations in Ca(2+) and Mg(2+) homeostasis and that CLDN16-deficient mice represent a useful model to further elucidate pathways involved in renal Ca(2+) and Mg(2+) handling.

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology.

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites.

Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.

Efficient modification of alpha-synuclein serine 129 by protein kinase CK1 requires phosphorylation of tyrosine 125 as a priming event.

S129-phosphorylated alpha-synuclein (α-syn) is abundantly found in Lewy-body inclusions of Parkinson's disease patients. Residues neighboring S129 include the α-syn tyrosine phosphorylation sites Y125, Y133, and Y136. Here, we use time-resolved NMR spectroscopy to delineate atomic resolution insights into the modification behaviors of different serine and tyrosine kinases targeting these sites and show that Y125 phosphorylation constitutes a necessary priming event for the efficient modification of S129 by CK1, both in reconstituted kinase reactions and mammalian cell lysates. These results suggest that α-syn Y125 phosphorylation augments S129 modification under physiological in vivo conditions.

Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts.

We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the ¹5N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D ¹H-¹5N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

Claudin-10 exists in six alternatively spliced isoforms that exhibit distinct localization and function.

The tight junction protein claudin-10 is known to exist in two isoforms, resulting from two alternative exons, 1a and 1b (Cldn10a, Cldn10b). Here, we identified and characterized another four claudin-10 splice variants in mouse and human. One (Cldn10a_v1) results from an alternative splice donor site, causing a deletion of the last 57 nucleotides of exon 1a. For each of these three variants one further splice variant was identified (Cldn10a_v2, Cldn10a_v3, Cldn10b_v1), lacking exon 4. When transfected into MDCK cells, Cldn10a, Cldn10a_v1 and Cldn10b were inserted into the tight junction, whereas isoforms of splice variants lacking exon 4 were retained in the endoplasmic reticulum. Cldn10a transfection into MDCK cells confirmed the previously described increase in paracellular anion permeability. Cldn10a_v1 transfection had no direct effect, but modulated Cldn10a-induced organic anion permeability. At variance with previous reports in MDCK-II cells, transfection of high-resistance MDCK-C7 cells with Cldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells.

Localization of the epithelial Ca(2+) channel in rabbit kidney and intestine.

The epithelial Ca(2+) channel (ECaC), which is exclusively expressed in 1,25-dihydroxyvitamin D(3)-responsive tissues, i.e., kidney, intestine, and placenta, is postulated to constitute the initial step in the process of transcellular Ca(2+) transport. To strengthen this postulated function, the present study compares the segmental and cellular distribution of ECaC and the other Ca(2+) transport proteins known to be involved in transcellular Ca(2+) transport. In rabbit kidney, ECaC mRNA and protein expression were primarily present in the connecting tubule. Immunopositive staining for the ECaC protein was exclusively found at the apical domain of this tubular segment. Importantly, ECaC completely colocalized with calbindin-D(28K), Na(+)-Ca(2+) exchanger (NCX), and plasma membrane Ca(2+) -ATPase (PMCA). A minority of cells along the distal tubule lacked immunopositive staining for ECaC and the other Ca(2+) transporting proteins. These negative cells were identified as intercalated cells. In intestine, ECaC was present in a thin layer along the apical membrane of the duodenal villus tip, whereas the crypt and goblet cells were negative. Again, a complete colocalization was observed between ECaC, calbindin-D(9K), and PMCA. In contrast to the kidney, NCX could not be detected in duodenum. The present finding that ECaC completely colocalizes with the Ca(2+) transport proteins in the connecting tubule and duodenum, together with its apical localization, further substantiates the postulated function of ECaC as the gatekeeper of active Ca(2+) (re)absorption.