A study of intrinsic disorder and its role in functional proteomics

Thesis (Ph.D.) - Indiana University, Informatics, 2009The last decade has witnessed the emergence of an alternate view on how protein function arises. This view attributes the functionality of many proteins to the presence of an ensemble of flexible regions popularly as `intrinsically disordered' or `unstructured'. Several proteomic studies have corroborated the existence of either wholly disordered proteins or proteins that contain regions of disorder in them. The purpose of this dissertation was to investigate the consistency of such regions across experiments, their mechanism of facilitating function via disorder-to-order transitions, their presence and significance in pathogenic versus non-pathogenic organisms and their promise of applicability towards the computational prediction of peptides involved in the most common class of post-translational modifications, phosphorylation. Besides these, a new algorithm exploiting the strong correlation between phosphorylation and intrinsic disorder has also been proposed to improve the detection of phosphorylated peptides via high-throughput methods such as tandem mass-spectrometry (LC-MS/MS). Results presented in this study, guide us in understanding the robustness of unstructured regions in proteins to sequence changes and environment, their role in facilitating molecular recognition as well as improving currently available methods for identification of post-translationally modified peptides. The findings and conclusions of this dissertation have the potential to impact ongoing structural genomics initiatives by suggesting alternative methods for determining structure for targets containing regions of disorder. Additional ramifications of results from this work include directing attention towards the possible use of regions of intrinsic disorder by pathogenic organisms for host cell invasion. We believe that unlike the traditional reductionist approach in a scientific method, this study gathers strength and utility by investigating the role of intrinsic disorder on more than one front in order to provide a novel perspective to the understanding of complex interactions within biological systems. Concluding arguments presented in this study pique one's curiosity regarding the evolution of disordered regions and proteins in general. On a technological side, the findings from this study unequivocally support the viable use of informatics methods in gaining new insights about a relatively young class of proteins known as intrinsically disordered proteins and its applicability to improve our present knowledge of cellular physiology

The role of N1-Src regulated splicing in neuronal differentiation

Alternative splicing (AS) is one of the main contributors to transcriptome diversity and functional complexity involved in the process of neuronal development. Evidence suggests that many splicing regulators and alternative splicing events are neuron-specific and aberrations in the regulation of these events have been linked to various neurodevelopmental disorders. N1-Src is an evolutionarily conserved neuronal splice variant of the ubiquitous tyrosine kinase c-Src. It has been implicated in neural development and as a prognostic indicator in neuroblastoma, a childhood cancer that is caused by failure of neural crest cells to differentiate. Results from knockdown experiments where N1 exon inclusion was prevented with splice-blocking antisense morpholino oligos revealed that N1-Src is a key regulator of primary neurogenesis in Xenopus. Preliminary short and long read RNA-Seq data from Xenopus embryos suggest a role for N1-Src in regulation of an alternative splicing programme during early neurogenesis, with transcripts encoding the splicing/RNA processing machinery themselves being the most spliced targets. This study aimed to further describe the N1-Src-regulated splicing network in the developing Xenopus nervous system using bioinformatic analysis of various publicly available and Evans/Isaacs lab RNASeq datasets. A differential splicing (DS) analysis pipeline was developed to detect and quantify alternative splicing events that occur during early stages of Xenopus embryo development relevant to neurogenesis. By correlating alternative splicing quantifications with RNA-binding protein motif enrichment analysis, this project proposed mechanisms for Src regulation of alternative splicing

Engineering Dynamic Behavior into Nucleic Acids Guided by Single Molecule Fluorescence Microscopy

Single-molecule fluorescence microscopy is a powerful technique that has been used for investigating the structural dynamics of biomolecules, and is particularly useful when ensemble averaging might obscure detailed information of the system under investigation. One application of single molecule measurement is to optimize the design of DNA nano-devices. Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation, and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or more to carry out a handful of operations. In this thesis, I have pursued the speed limit of an important class of reactions in DNA nanotechnology—toehold exchange—through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. I identified the walking mechanism by single-molecule fluorescence resonance energy transfer (smFRET) measurement, with the stepping rate constant approaching 1 s-1, which is 10- to 100-fold faster than prior DNA walkers. I also used single-particle tracking to demonstrate movement of the walker over hundreds of nanometers within 10 min, in quantitative agreement with predictions from the stepping kinetics. These results suggest that substantial improvements in the operating rates of broad classes of DNA nanomachines utilizing strand displacement are possible. Another application of single molecule measurements is kinetic fingerprinting detection. Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this thesis, I review important experimental tips and data analysis details to provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-base selectivity by monitoring the repetitive interactions of fluorescent probes with immobilized targets. In addition to demonstrating how this kinetic fingerprinting filters out background arising from the inevitable nonspecific binding of probes, yielding virtually zero background signal, I also investigated the detection of epigenetic mutations such as CpG methylation using SiMREPS. The analysis of single-molecule microscopy data can be very time-consuming because there is no sufficiently robust automatic method for selection of qualified single-molecule fluorescence trajectories from the generally noisy and heterogeneous raw data, necessitating manual trace selection that can take hundreds of hours for large datasets. In this thesis, I discuss the innovative use of the popular convolutional neural network AlexNet and the recurrent neural network Long Short-Term Memory (LSTM) to develop an automatic selector for single-molecule fluorescence resonance energy transfer (smFRET) traces. The average prediction accuracy is above 90% when tested on datasets from different users and experimental systems. To boost the selection accuracy and increase the diversity of training datasets, simulation data were included into the training data set and tested for selection accuracy. I expect that this new method will not only greatly expedite analysis of smFRET data and increase analysis reliability of SiMREPS data, but also introduce and validate machine learning as an effective tool for analysis of single-molecule microscopy data more generally. Together, these results provide new insights into how single molecule microscopy can be used to engineer dynamic behaviors of nucleic acids.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149970/1/jmli_1.pd

A METABOLOMIC, STRUCTURAL AND FUNCTIONAL STUDY OF POLYCYSTIN1 AND NEPHROCYSTIN1

Abstract Nephronophthisis (NPHP) and Autosomal Dominant Polycystic Kidney Disease (ADPKD) are two genetic renal cystic diseases that lead to End Stage Renal Disease (ESRD) in childhood or adolescence and at the late-middle age, respectively1. At present the mechanisms at the basis of cystogenesis in both diseases are poorly understood and pharmacological therapies are still lacking. On the one hand NPHP is characterized by kidney tubular atrophy and cysts formation occurring primarily at the cortico-medullary border, that leads to a reduction of kidneys volume. It is caused by mutations in 11 different genes. The most common form of NPHP is the juvenile one (NPHP type 1), which is caused, in most of the cases, by homozygous deletion of NPHP1 gene (2q13 chromosome). NPHP1 gene encodes for nephrocystin1 (NPHP1), a 732 amino acids long cytoplasmatic protein. It presents a widespread expression, but the pathogenic consequences due to loss-of-function are mainly confined to the kidney. In particular, NPHP1 is localized to cell-cell junctions, cilia and cell-matrix adhesion sites and it is involved in signaling transduction, cell-cell adhesion and cell polarity2-4. On the other hand the hallmark of ADPKD is the formation of bilateral cysts that leads to an increase of kidneys volume weight5,6. The disease is caused by mutations in two genes: Pkd1 gene (16p13.3 chromosome) which is mutated in 85% of cases and Pkd2 (4q21 chromosome) which is mutated in the remaining part of cases. These genes encode respectively for polycystin1 (PC1), a large plasma membrane receptor 4302 amino acids long and polycystin2 (PC2), a 968 amino acids long calcium channel. The two proteins interact through their C-terminal coiled coil domains and are involved in common signaling pathways, thus explaining the same phenotype5,7,8. In this thesis we focused our attention on two new possible roles of PC1: the regulation of metabolism and its interaction with NPHP1. To disentangle PC1 role as \u201cmetabolic regulator\u201d and its possible interaction with NPHP1 through its coiled coil domain, we have selected nuclear magnetic resonance (NMR) spectroscopy as primary investigation technique. PC1 as metabolic regulator. During a routine culture of mouse embryonic fibroblasts (MEFs) our collaborators (Dr. Boletta\u2019s group in San Raffaele Hospital, Milan, Italy) have observed that the knock out (Pkd1 KO) MEFs for PC1 acidify the medium faster than the wild type (WT) ones. This finding rises the hypothesis that PC1 is involved in the regulation of cellular metabolism. Hence, exploiting nuclear magnetic resonance (NMR) spectroscopy, we have applied a metabolomic approach in order to investigate the metabolic pathways and the signaling cascade deregulated by the loss of PC1. Importantly, the metabolomic analysis was performed both in cells and in mouse kidneys (WT and Pkd1 KO) in order to compare the results both in vitro and in vivo. The analysis of the 1D 1H NMR spectra of the media conditioned by the Pkd1 KO and by the WT MEF cells (exometabolome) has highlighted some metabolic differences: the exometabolome of the Pkd1 KO MEF cells displays an increased content of lactate, glutamate and alanine, presenting features similar to tumor cell metabolism9-11whereas the exometabolome of the WT MEF cells has an increased content of choline, formate, glucose, glycine and pyruvate. The two metabolites that vary mostly between the two cell lines are glucose and lactate, as highlighted by PCA analysis. In particular Pkd1 KO MEF cells display a major consumption of glucose, which is in turn related to a major production of lactate, showing the aerobic glycolysis, also called Warburg effect, a metabolic hallmark usually observed in cancer cells. In order to prove the occurrence of the Warburg effect in vivo, Ksp-Cre: Pkd1flox/- and Pkd1flox/+ mice12 were treated with uniformly labeled 13C glucose13,14. 1H-13C HSQC NMR spectra on the polar extracts of kidneys and livers (used as control organ) were acquired and analyzed in order to simplify the detection of the metabolic conversion of glucose into lactate. The Pkd1 KO kidneys displayed an increased content of glucose and lactate as compared to the WT ones, whereas livers did not display any difference. These results confirm that the aerobic glycolysis occurs also in vivo and is caused by the loss of functional PC1. In order to restore the metabolic basal condition, mice were treated with 2-deoxy-glucose (2DG), a drug able to block directly the glucose metabolism15,16. For testing the efficacy of the treatment, Ksp-Cre: Pkd1flox/- and Pkd1flox/+ mice were treated at the same time with 13C glucose and 2DG and the polar extracts of kidneys and livers were analyzed through 1H-13C HSQC NMR spectra. The Pkd1 KO kidneys treated with 2DG display a decrease of the lactate content, whereas the amount of glucose does not change, hence the treatment with 2DG affects the glucose metabolism but not its uptake. Moreover, from the histological point of view, the treated Pkd1 KO kidneys show a reduction of the volume of cysts and consequently a reduction of the weight of the kidney, compared to the not treated Pkd1 KO kidneys. Pkd1 KO livers do not present any changes after treatment with 2DG. These data show that 2DG acts specifically on Pkd1 KO kidneys, improving both the altered metabolism and the histology of the Pkd1 KO kidneys, without involving other organs that, instead, express functional PC1 (eg. the liver). In conclusion, our findings indicate that PC1 acts as \u201cmetabolic regulator\u201d, as shown by the analysis of the medium conditioned Pkd1 KO cells. In particular Pkd1 KO cells and Pkd1 KO kidneys are affected by the Warburg effect, a metabolic hallmark of accelerated metabolism observed also in cancer cells. Moreover, the analysis of Pkd1 KO kidneys of Ksp-Cre: Pkd1flox/- and Pkd1flox/+ mice treated with 2DG has highlighted an improvement of the Warburg effect and the renal histology. These preliminary results are very promising for a possible therapy for ADPKD patients and will be object of further in vivo studies. The coiled coil domain of PC1 does not interact with the N-terminus of NPHP1. We have recently demonstrated that one of the poly-proline motives, belonging to cytoplasmic C-terminus of PC1, is a binding partner for SH3 domain of NPHP1 and this complex is involved in the regulation of apoptosis17. The weak affinity of the resulting complex, as indicated by the estimated dissociation constant (Kd=0.3 +/-0.02 mM), suggests that other proteins or other domains of the same proteins could contribute to the interaction. In particular, the presence of coiled coil domains at PC1 C-terminus and at NPHP1 N-terminus, suggests the formation of a more complex macromolecular system involving interactions of both coiled coils. In order to verify and to structurally characterize at atomic level the interactions between PC1 and NPHP1 we have applied NMR spectroscopy. The first 115 amino acids (13kDa) of NPHP1 were predicted to form a coiled domain by the Web Server PCOIL. The coiled coil domain is a \u3b1 helical motif involved in homo and heteromultimerization18. Unexpectedly, recombinant NPHP11-115 behaves as a monomer, as assessed by the elution volume in size exclusion chromatography and by the overall correlation time deduced from NMR relaxation experiments. Circular dichroism experiments confirmed the presence of \u334 45% of \u3b1 helix and indicated a high thermostability of the domain, with a melting temperature of 65\ub0C. The solution structure of the domain shows that the first 115 amino acids of NPHP1 present a three helix bundle fold stabilized by hydrophobic interactions. The supposed interaction between the N-terminus of NPHP1 and the coiled coil domain of PC1 (CC_PC1) was next verified performing an NMR titration with the purified recombinant proteins, recording 1H-15N HSQC NMR spectra of NPHP1 upon addition of unlabelled CC-PC1. CC_PC1 was produced with the tags in order to improve the solubility and to avoid aggregation19. The recorded 1H -15N HSQC spectrum does not show any chemical shift displacement, indicating, that the two proteins do not interact. In conclusion our data show that the N-terminus of NPHP1 is a monomeric and thermostable domain characterized by the presence of three \u3b1 helices that pack together to form a three helix bundle. The N-terminus of NPHP1 is not a coiled coil domain, as erroneously predicted, and does not interact with CC_PC1. Interactions between NPHP1-115 and PC1 should be modulated by other protein domains or might involve additional proteins. Further experiments are needed to clarify this point

Understanding the Organization and functional Control of Polysomes by integrative Approaches

Background and rationale Translation is a fundamental biological process occurring in cells, carried out by ribosomes simultaneously bound to an mRNA molecule (polyribosomes). It has been exhaustively demonstrated that dysregulation of translation is implicated in a wide collection of pathologies including tumours and neurological disorders. Latest findings reveal the existence of translational regulatory mechanisms acting in cis or trans with respect to the mRNAs and governing the movement and the position of ribosomes along transcripts or directly impacting on the ribosome catalogue of its constituent proteins. For this reason, translational controls also account for widespread uncoupling between transcript and protein abundances in cells. To explain the poor correlation between transcripts and protein levels, many computational models of translation have been developed. Usually, these approaches aim at predicting protein abundances in cells starting from the mRNA abundance. Despite the efforts of these modelling studies, a consensus model remains elusive, drawing to contradictory conclusions concerning the role of mRNA regulatory elements such as the usage of codons (codon usage bias) and slowdown mechanism at the beginning of the coding sequence (ramp). More recently, following the rapid and widespread diffusion of ribosome footprinting assays (RiboSeq), which enables the dissection of translation at single nucleotide resolution, a number of computational pipelines dedicated to the analysis of RiboSeq data have been proposed. These tools are typically designed for extracting gene expression alterations at the translational level, while the positional information describing fluxes and positions of ribosomes along the transcript is still underutilized. Therefore, the polysome organization, in term of number and position of ribosomes along the transcript and the translational controls directed in shaping cellular phenotypes is still open to breakthrough discoveries. Broad objectives The aim of my thesis is the development of mathematical and computational tools integrated with experimental data for a comprehensive understanding of translation regulation and polysome organization rules governing the number of ribosomes per polysome and the ribosome position along transcripts. Project design and methods With this purpose, I developed riboWaves, an integrated bioinformatics suite divided in two branches. riboWaves includes in the first branch two modeling modules: riboAbacus, predicting the number of ribosomes per transcript, and riboSim, predicting ribosome localization along mRNAs. In the second branch, riboWaves provides two pipelines, riboWaltz and riboScan, for detailed analyses of ribosome profiling data aimed at providing meaningful and yet unexplored ribosome positional information. The models and the pipelines are implemented in C and R, respectively. riboAbacus and riboWaltz are available on GitHub. Results To predict the number of ribosomes per transcript and the position of ribosomes on mRNAs, I applied riboAbacus and riboSim, respectively, to transcriptomes of different organisms (yeast, mouse, human) for understanding the role of translational regulatory elements in tuning polysome in different organisms. First, I trained and validated performances of riboAbacus taking advantage of Atomic Force Microscopy images of polysomes, while performances of riboSim were assessed employing ribosome profiling data. Predictions provided by riboAbacus and riboSim were evaluated in parallel. I showed that the average number of ribosomes translating a molecule of mRNA can be well explained by the deterministic model, riboAbacus, that includes as features the mRNA levels, the mRNA sequences, the codon usage bias and a slowdown mechanism at the beginning of the CDS (ramp hypothesis). The predictions of ribosome localization by riboSim that used as features the mRNA sequence, the codon usage and the ramp, were run for yeast, mouse and human. I observed a good similarity between the predicted and experimental positions of ribosomes along transcripts in yeast, while poor similarity was obtained between predicted and experimental ribosome positions in the two mammals, suggesting the presence of more elaborate controls that tune ribosomes movement in higher eukaryotes than in simple species. After having developed two tools for the analyses of RiboSeq data and extraction of positional information on ribosome localization along transcripts, I applied both riboWaltz and riboScan in a case study. The aim was to dissect possible defects in ribosome localization in tissues of a mouse model of Spinal Muscular Atrophy (SMA). SMA is a neurodegenerative disorder caused by low levels of the Survival of Motor Neuron protein (SMN) in which translational impairments are recently emerging as possible cause of the disease. I analysed ribosome profiling data obtained from three different types of RiboSeq variants in healthy and SMA-affected mouse brains at the early-symptomatic stage of the disease. I observed i) a significant drop-off of translating ribosomes along the coding sequence in the SMA condition (using riboWaltz); ii) in SMA-affected mice, the possible accumulation of ribosomes along the 3' UTR in neuro-related mRNAs (using riboScan); iii) the involvement of SMN-specialized ribosomes in playing a very intimate role with the elongation stage of translation of the first codons of transcripts (riboWaltz), iv) the loss of ribosomes at the 3rd codon in SMA in transcripts bound by SMN-specialized ribosomes and v) a remarkable connection between SMN and the down-regulation of genes in SMA-affected mice. Overall, these findings confirmed previous observation about possible SMN-related dysregulations of local protein synthesis in neurons. More importantly, they unravel a completely new role of SMN in tuning translation at multiple levels (initiation, elongation and the recycling of terminating ribosomes), opening new hypotheses and scenarios for explaining the most devastating genetic disease, leading cause worldwide of infant mortality. Conclusions The present work provides a new comprehensive and integrated scenario for better understanding translation and demonstrates that this approach is a very powerful strategy to pave the way for new understanding of fine alteration in polysome organization and functional control in both physiological and pathological conditions

Genetic variation in DNA repair proteins modifies the course of Huntington’s disease

Huntington’s disease (HD) is caused by a CAG repeat expansion in HTT on chromosome 4. Onset and progression are inversely correlated with repeat length, but a significant proportion of the variability in each is due to modifiers elsewhere in the genome. Recent genome-wide association studies have identified the DNA repair genes FAN1 and MSH3 as modifiers of onset and progression respectively. This thesis finds that variants associated with HD disease course also influence onset in other polyglutamine diseases, suggesting a shared pathogenic mechanism involving DNA repair. In blood from HD patients there is significant transcriptional dysregulation, particularly involving immune, metabolic and DNA repair pathways, which correlates with disease severity, parallels dysregulation seen in the most affected HD brain regions and overlaps with Alzheimer’s disease. To study the role of DNA repair, several cell models of somatic instability were developed, including patient-derived lymphoblasts and induced pluripotent stem cells, which show exponential repeat expansion that continues in differentiated medium spiny neurons (MSNs). In a FAN1 knockout U20S cell model of HD, FAN1 is shown to protect against repeat instability, and this function is dependent on protein concentration and CAG repeat length, but does not require its nuclease activity. shRNA-mediated FAN1 knockdown accelerates repeat expansion in both patient-derived iPSCs and MSNs. Through chromatin immunoprecipitation, FAN1 is shown to bind, but not specifically target, CAG repeat DNA. AAV9-mediated miRNA Fan1 knockdown in the striatum and liver of R6/2 mice did not accelerate repeat expansion, likely because only 23% knockdown was achieved. Illumina sequencing of the MSH3 region that influences HD progression identified a repeat variant that is associated with decreased MSH3 expression, reduced somatic expansion, delayed onset and slower progression in HD and myotonic dystrophy type 1 (DM1). These results suggest MSH3 promotes and FAN1 protects against repeat instability, which in turn influences the course of repeat expansion diseases

SORLA in renal and adrenal function

Der Typ I Transmembran-Rezeptor SORLA gehört zur VPS10p-Rezeptor Familie in Säugern. Der Rezeptor mit starker Homologie zu Endozytose- und Sorting-Rezeptoren ist am stärksten im zentralen Nervensystem (CNS) exprimiert. Außerhalb des CNS ist SORLA in einer Vielzahl von Geweben zu finden, unter anderem in der Niere. Das klare Expressionsmuster des Rezeptors im distalen Nephron lässt eine Rolle des Rezeptors in transepithelialen Transportprozessen vermuten. Um genau festzustellen, welche Prozesse von SORLA beeinflusst werden, wurde die Nierenfunktion von Mäusen mit einer vollständigen Defizienz des Sorla-Gens (Sorla-/-) untersucht. Diese Tiere zeigen Defekte in der renalen Ionenhomöostase: sie verlieren Na+, Cl-, K+, und Ca2+ (im Normalzustand und/oder nach Trinkwasserentzug). Eine Erniedrigung von Blutdruck und Herzfrequenz sowie eine fehlregulierte Sekretion von Aldosteron gehen mit dem Salzverlust-Phänotyp einher. Passend zu dieser Beobachtung konnte eine Expression von SORLA in der Nebenniere – speziell in der Zona glomerulosa, dem Ort der Aldosteron-Synthese – gezeigt werden. Des weiteren wurde eine signifikant verminderte Expression mehrerer Gene des Adrenalin-Synthesewegs in Sorla-/--Mäusen festgestellt, welcher in einer verringerten Menge des Hormons in den Nebennieren der Tiere resultiert. In der Niere bewirkt das Fehlen von SORLA insbesondere eine veränderte Phosphorylierung der beiden Kation-Chlorid-Cotransporter NCC und NKCC2 hervor, deren Aktivität durch Phosphorylierung reguliert wird. Es ist bekannt, dass die Signalkinase SPAK die Aktivität von NKCC2 und NCC reguliert. Die anormale Phosphorylierung fällt mit einer untypischen Verteilung der Kinase im TAL der SORLA-defizienten Mäuse zusammen. Dies deutet auf eine Funktion des Rezeptors beim Trafficking von SPAK hin. Durch die Identifizierung von Transportproteinen als putative Interaktionspartner SORLAs konnte diese Hypothese bekräftigt werden.The type I transmembrane receptor SORLA is a member of the mammalian VPS10p-receptor family. The receptor, which is mainly expressed in the central nervous system (CNS), is characterized by high structural homology to endocytosis- and sorting-receptors. Outside the CNS, expression of SORLA can be found in a variety of tissues, including kidney. This distinct expression pattern in the distal nephron suggests a role for SORLA in transepithelial transport processes. To determine which processes the receptor might be involved in, the kidney function of mice, wich carry a complete deficiency of the Sorla gene, was analyzed. These animals show defects in ion handling: they are wasting Na+, Cl-, K+, and Ca2+ (under normal conditions and/or after water deprivation). The salt loss phenotype is accompanied by decreased mean arterial pressure and heart rate as well as mis-regulated secretion of aldosterone. In line with this observation, SORLA is also expressed in the adrenal gland, particularly in the zona glomerulosa, the place of aldosterone synthesis. Additionally, a significant down-regulation of several genes of the epinephrine synthesis pathway in mice lacking SORLA was found. This defect results in lower adrenal levels of the hormone. In the kidney, the lack of SORLA results especially in altered phosphorylation of the two cation-chloride cotransporters NCC and NKCC2, as their activity is regulated by phosphorylation. The signaling kinase SPAK has been reported to regulate the activity of NKCC2 and NCC. The transporters’ abnormal phosphorylation coincides with the atypical distribution of the kinase in TAL of Sorla-/- mice, suggesting a role of the receptor in establishing the localization of SPAK. This hypothesis was further substantiated by the identification of putative SORLA-interacting proteins involved in trafficking