Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity.

Protein arginine methylation plays a critical role in differential gene expression through modulating protein-protein and protein-DNA/RNA interactions. Although numerous proteins undergo arginine methylation, only limited information is available on how protein arginine methyltransferases (PRMTs) identify their substrates. The human PRMT5 complex consists of PRMT5, WD45/MEP50 (WD repeat domain 45/methylosome protein 50), and pICln and catalyzes the symmetrical arginine dimethylation of its substrate proteins. pICln recruits the spliceosomal Sm proteins to the PRMT5 complex for methylation, which allows their subsequent loading onto snRNA to form small nuclear ribonucleoproteins. To understand how the PRMT5 complex is regulated, we investigated its biochemical composition and identified RioK1 as a novel, stoichiometric component of the PRMT5 complex. We show that RioK1 and pICln bind to PRMT5 in a mutually exclusive fashion. This results in a PRMT5-WD45/MEP50 core structure that either associates with pICln or RioK1 in distinct complexes. Furthermore, we show that RioK1 functions in analogy to pICln as an adapter protein by recruiting the RNA-binding protein nucleolin to the PRMT5 complex for its symmetrical methylation. The exclusive interaction of PRMT5 with either pICln or RioK1 thus provides the first mechanistic insight into how a methyltransferase can distinguish between its substrate proteins.

A stimulatory role for the La-related protein 4B in translation.

La-related proteins (LARPs) belong to an evolutionarily conserved family of factors with predicted roles in RNA metabolism. Here, we have analyzed the cellular interactions and function of LARP4B, a thus far uncharacterized member of the LARP family. We show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interaction of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for activated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditions, LARP4B co-sedimented with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, overexpression of LARP4B stimulated protein synthesis, whereas knockdown of the factor by RNA interference impaired translation of a large number of cellular mRNAs. In sum, we identified LARP4B as a stimulatory factor of translation. We speculate that LARP4B exerts its function by bridging mRNA factors of the 3' end with initiating ribosomes.

A qualitative study on patients' and physicians' visions for the future management of overweight or obesity.

BACKGROUND: The management of obesity with its associated morbidity and mortality is a growing problem in primary care practices. Despite numerous recommendations in response to this challenge, weight management interventions still yield poor results. This is partly due to a discrepancy between physicians' and patients' understanding of the problem and possible solutions. OBJECTIVE: This study analyses patients' and physicians' visions for the future management of obesity. METHODS: Qualitative in-depth semi-structured interviews were performed. Physicians and patients were asked about their individual needs, experience and views regarding the management of obesity. Fifteen GPs and 15 overweight patients participated in this study. Interviews were transcribed and submitted to qualitative content analysis. RESULTS: The investigation reveals a high level of agreement between the two groups with regard to communication requirements for successful care. Both groups stressed the need for multimodal care concepts inside and outside of GP practices. Both also addressed the current overburdening of outpatient care structures in dealing with the management of obesity. CONCLUSION: Options should be developed for closer cooperation between GPs and support facilities inside and outside practices.

Evolution of an RNP assembly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster.

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2-8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. Our data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction.

The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and affects transcription of cellular and viral polymerase II genes.

The positive transcription elongation factor b (P-TEFb) is a heterodimeric complex composed of cyclin-dependent kinase 9 and its regulator cyclin T1/2. It stimulates transcription elongation by phosphorylation of serine 2 residues in the carboxy-terminal domain of polymerase II. 7SK RNA and HEXIM proteins can antagonize transcriptional stimulation by sequestering P-TEFb in a catalytically inactive ribonucleoprotein (RNP). Here, we show that the previously uncharacterized La-related protein 7 (LARP7) has a role in 7SK-mediated regulation of transcription. LARP7 binds to the highly conserved 3'-terminal U-rich stretch of 7SK RNA and is an integral part of the 7SK RNP. On stimulation, LARP7 remains associated with 7SK RNA, whereas P-TEFb is released. Interestingly, reduction of LARP7 by RNA interference enhances transcription from cellular polymerase II promoters, as well as a TAT-dependent HIV-1 promoter. Thus, LARP7 is a negative transcriptional regulator of polymerase II genes, acting by means of the 7SK RNP system.

Application of electron transfer dissociation (ETD) for the analysis of posttranslational modifications.

Despite major advantages in the field of proteomics, the analysis of PTMs still poses a major challenge; thus far, preventing insights into the role and regulation of protein networks. Additionally, top-down sequencing of proteins is another powerful approach to reveal comprehensive information for biological function. A commonly used fragmentation technique in MS-based peptide sequencing is CID. As CID often fails in PTM-analysis and performs best on doubly-charged, short and middle-sized peptides, confident peptide identification may be hampered. A newly developed fragmentation technique, namely electron transfer dissociation (ETD), supports both, PTM- and top-down analysis, and generally results in more confident identification of long, highly charged or modified peptides. The following review presents the theoretical background of ETD and its technical implementation in mass analyzers. Furthermore, current improvements of ETD and approaches for the PTM-analysis and top-down sequencing are introduced. Alternating both fragmentation techniques, ETD and CID, increases the amount of information derived from peptide fragmentation, thereby enhancing both, peptide sequence coverage and the confidence of peptide and protein identification.

Motivational interviewing and shared decision making in primary care.

OBJECTIVE: The aim of this study was to assess general practitioners' (GP) readiness to involve obese patients in therapy decision making and to determine whether they integrate motivational interviewing techniques. METHODS: Fifty-eight preventive Check-up 35 encounters with overweight and obese patients in primary care were audio recorded in 12 GP practices. The use of motivational interviewing techniques was rated with the Behavior Change Counseling Index (BECCI). The involvement in medical decisions was rated with the Observing Patient Involvement Scale (OPTION). RESULTS: OPTION and BECCI scores were low (means=0.71 and 1.65), indicating minimal implementation of shared decision making and motivational interviewing in preventive encounters with these patients. GPs used more motivational interviewing for patients with a BMI>30 kg/m(2) than for those with a BMI<30 kg/m(2). Female GPs had significantly higher shared decision making scores, indicating that they prefer to involve patients in medical decisions. GPs differed significantly in their use of both approaches. CONCLUSIONS: Shared decision making and motivational interviewing, though known to be successful strategies in lifestyle counseling, are rarely used during obesity encounters in our sample of German GPs. PRACTICE IMPLICATIONS: GPs should be sensitized and trained in the application of these methods.

Counseling overweight in primary care: an analysis of patient-physician encounters.

OBJECTIVE: The aim of this study was to assess general practitioners' (GPs') and patients' practices and attitudes regarding overweight encountered during preventive counseling talks. METHODS: Twelve GPs audiotaped their preventive counseling talks with overweight patients, including the assessment of individual risk profiles and further medical recommendations. Fifty-two dialogues were transcribed and submitted to qualitative content analysis. RESULTS: Dietary advice and increased physical activity are mostly discussed during talks. Recommendations appear to be more individual if patients are given the chance to reflect on causes of their overweight during counseling talks. CONCLUSIONS: A dialogue approach affects the strength and quality of weight loss counseling in primary care. However, physicians and overweight patients rarely agreed on weight loss goals during the physician-patient talks. PRACTICAL IMPLICATIONS: Patient centeredness, particularly the integration of patients' perceptions towards weight management, might be an important step towards improving weight counseling in primary care.

[Overweight patients in primary care: how family physicians utilize medical check-up examinations for risk counselling. Content analysis of check-up counselling talks between physicians and patients]. / Ubergewichtige Patienten in der Hausarzt- praxis: Wie wird die Gesundheitsuntersuchung zur Risikoberatung genutzt? Eine qualitative Analyse von Arzt-Patient-Gesprächen.

BACKGROUND: The number of overweight patients in Germany has been continuously increasing during the past years. The so-called "check-up 35" consultation presents primary care providers with an opportunity for preventive counselling talks with the respective individuals. In this qualitative study we analysed family physician encounters. We were particularly interested in the way in which the subject matter was addressed and the risk counselling performed by the physicians when confronted with overweight patients. METHODS: Twelve physicians audio-taped their final check-up dialogue with 52 overweight or obese patients (BMI > or = 25 kg/m2). The interviews were transcribed, and a content analysis was conducted using the established method developed by Mayring. RESULTS: Physicians used direct or more often indirect strategies to address overweight in counselling talks that were most often initiated by their patients. They largely addressed this topic while communicating laboratory results. Some obese patients did not receive any advice on health risks. In the encounters analysed the physicians did not employ standardized risk counselling tools. CONCLUSIONS: The reasons for addressing overweight indirectly and the lack of standardized risk counselling with obese patients are carefully discussed.

Native purification of protein and RNA-protein complexes using a novel affinity procedure.

Genetic studies in invertebrate model organisms such as Drosophila melanogaster have been a fundament of cell and developmental biology for more than one century. It is mainly the lack of an efficient purification strategy which has hampered biochemical and proteomic analyses of gene products. We describe a novel affinity-tag, termed TagIt-epitope, specifically designed for affinity-purifications of multiprotein complexes from Drosophila. TagIt-fusion proteins can be efficiently purified using a monoclonal antibody and eluted under native conditions by competition with synthetic peptide encompassing the epitope. We demonstrate that this tag is suitable for the purification of proteinaceous assemblies such as the PRMT5-complex and RNA-protein complexes such as snoRNPs from Drosophila Schneider2 cells. Furthermore, we describe a novel approach by which this tag can be used to affinity-purify RNA-binding proteins from cell extracts. Therefore, the TagIt-technique or modifications thereof will be of great value in analyzing macromolecular complexes in Drosophila and also other invertebrates by biochemical means. In addition, RNA-peptide hybrid molecules may become a novel tool to purify RNA binding proteins.

Phosphoproteome of resting human platelets.

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.