Epidemiology and Treatment of Patients With Primary Open Angle Glaucoma in Germany: A Health Claims Data Analysis.

PRCIS: Based on a large administrative database of German claims data, our study provides current estimates of the prevalence and incidence of primary open-angle glaucoma (POAG) in Germany and describes selected outcomes for prevalent POAG patients. PURPOSE: To estimate the prevalence and incidence of POAG in Germany, to describe the patient population in terms of comorbidity burden, routine care, and overall healthcare resource utilization (HCRU) and associated costs, and to describe treatment patterns over time in patients undergoing relevant laser procedures. MATERIALS AND METHODS: Based on anonymized German claims data, we carried out a retrospective, non-interventional study covering calendar years 2016 to 2021. RESULTS: For the adult German population (≥18 y), we estimated a POAG one-year prevalence of 1.70% and a one-year incidence of 0.17% in 2018; both increased with age, peaking in 80-89 year-olds. Prevalence and incidence were lower in 2020 (1.65% and 0.16%, respectively), the first year of the SARS-CoV-2 pandemic. Most patients solely received topical treatment. Most surgically-treated patients underwent laser trabeculoplasty, followed by laser iridotomy, trabeculectomy, and filtration operations with implant. In patients undergoing laser trabeculoplasty, the treatment regimen was nearly unchanged in the second year after, compared to two years before the procedure. Multimorbidity was commonly observed; 75.5% of patients had arterial hypertension and 50.0% had disorders of lipoprotein metabolism and other lipidemias, compared to 60.1% and 39.2%, respectively, in an age- and sex-matched control sample. CONCLUSIONS: Our study provides insights into epidemiology and routine care of POAG in Germany and HCRU in prevalent patients. There was little change in treatment regimens in patients who underwent laser trabeculoplasty, two years after the procedure. Most patients were multimorbid highlighting the need for comprehensive care.

Epidemiology and Real-World Treatment of Incident Diffuse Large B-cell Lymphoma (DLBCL): A German Claims Data Analysis.

INTRODUCTION: The objective of this study was to investigate the prevalence, incidence, and treatment patterns (treatment regimens, switches, duration) for diffuse large B-cell lymphoma (DLBCL) in a real-world setting. METHODS: This was a retrospective German claims data analysis of patients with DLBCL diagnosed between January 1, 2012, and December 31, 2020. The prevalence and cumulative incidence of DLBCL were found for 2019/2020. Line of treatment  (LOT) and treatment setting from first DLBCL diagnosis to end of follow-up were described. Kaplan-Meier overall survival (OS) estimates since DLBCL diagnosis and start of treatment lines were calculated. RESULTS: Overall, 2633 incident DLBCL cases were identified (median age 75 years, 51% male). Of these, 2119 patients received at least one DLBCL-related treatment (LOT1), and 1567 patients died during follow-up. In 2019/2020, the prevalence and cumulative incidence of DLBCL was 34.8/36.7 per 100,000 patients and 14.0/12.7 per 100,000 patients, respectively. For LOT1, 1922 patients were given a chemotherapy-based regimen (1530 with CD20 antibodies). A total of 403 patients were administered a second line (LOT2), of which 183 patients received a CD20 antibody-containing chemotherapy regimen and 100 patients received stem cell transplantation or chimeric antigen receptor (CAR)-T therapy. Of the 136 LOT3+ treatments, 74 were chemotherapy regimens (54 with CD20 antibodies) and 18 were kinase inhibitors. The median time between treatment lines was less than 6 months. Among patients with at least LOT2, approximately 50% received more than one LOT during the first year after diagnosis. Approximately 25% of treated patients died within 6 months of treatment initiation. Of the 2633 included patients, the median OS from diagnosis was 31.0 months (treated patients: 46.8 months, untreated patients: 3.0 months). CONCLUSIONS: Despite advances in the field, high unmet medical need in DLBCL remains. The treatment landscape is very heterogeneous, particularly in second- or later-line treatments, with few patients receiving potentially curative treatment beyond the first line. Treatment for DLBCL, particularly for transplant-ineligible patients, remains challenging.

Real-world treatment patterns and healthcare resource utilization among migraine patients: a German claims database analysis.

AIMS: Despite migraine being one of the most common neurological diseases, affected patients are often not effectively treated. This analysis describes the burden of migraine in Germany and assesses real-world treatment patterns and healthcare resource utilization (HCRU) of preventive-treated migraine patients from the perspective of Statutory Health Insurance. METHODS: A retrospective analysis was conducted using InGef Research Database claims data from 2018-2019. Migraine patients were stratified into cohorts by acute and preventive treatment status. Patients on preventive treatment were further stratified according to the type of prophylaxis received. Disease burden in preventively treated migraine patients was reported via treatment patterns, pathways, and comorbidities. HCRU was assessed through outpatient provider visits, hospitalizations, and sick leave. RESULTS: 160,164 adult migraine patients were identified, of which 55,378 (34.6%) were prescribed preventive treatment with conventional (n = 25,984, 46.9%), calcitonin gene-related peptide monoclonal antibody (CGRP mAb) (n = 613, 1.1%), or off-label therapies (n = 28,781, 52.0%). 936 (1.7%) patients received Botulinum Neurotoxin Type A (BoNTA). CGRP mAb-treated patients had a high rate of triptan prescriptions (2018: 95.5%; 2019: 88.9%), migraine-related hospitalizations (2018: 33.0%; 2019: 21.0%), and sick leave (2018: 26.8%; 2019: 22.5%). A high proportion of CGRP mAb- and BoNTA-treated patients was diagnosed with abdominal and pelvic pain (34.3% and 36.2%) and low back pain (34.1% and 35.3%). These patients also showed a high prevalence of depressive episodes (49.1% and 50.1%) and chronic pain disorders (37.5% and 32.9%). LIMITATIONS: This study focused on descriptive analyses which do not allow for assessment of causality when comparing treatment groups. CONCLUSIONS: Disease burden was high in patients receiving CGRP mAbs suggesting that patients treated preventively with CGRP mAbs shortly after product launch in Germany were severely affected, chronic migraine patients. The same may be true for patients receiving BoNTA who also showed an increased disease burden.

Structure of McsB, a protein kinase for regulated arginine phosphorylation.

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.

Structural basis for the disaggregase activity and regulation of Hsp104.

The Hsp104 disaggregase is a two-ring ATPase machine that rescues various forms of non-native proteins including the highly resistant amyloid fibers. The structural-mechanistic underpinnings of how the recovery of toxic protein aggregates is promoted and how this potent unfolding activity is prevented from doing collateral damage to cellular proteins are not well understood. Here, we present structural and biochemical data revealing the organization of Hsp104 from Chaetomium thermophilum at 3.7 Å resolution. We show that the coiled-coil domains encircling the disaggregase constitute a 'restraint mask' that sterically controls the mobility and thus the unfolding activity of the ATPase modules. In addition, we identify a mechanical linkage that coordinates the activity of the two ATPase rings and accounts for the high unfolding potential of Hsp104. Based on these findings, we propose a general model for how Hsp104 and related chaperones operate and are kept under control until recruited to appropriate substrates.

Arginine phosphorylation marks proteins for degradation by a Clp protease.

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC-ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC-ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC-ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin-proteasome system.

Versatile in vitro system to study translocation and functional integration of bacterial outer membrane proteins.

Gram-negative bacteria use the type-V secretion pathway to expose proteins at their cell surface, many of which have virulence functions. Translocation of those proteins across the outer membrane occurs either by means of dedicated translocator proteins (two-partner secretion) or covalently fused translocator domains (autotransporters). Translocator proteins and translocator domains are ß-barrels requiring the ß-barrel assembly machinery (BAM) for membrane integration. However, the molecular details of their passage across the envelope and insertion into the outer membrane remain enigmatic, owing in part to the fact that in vitro systems are not available. Here we describe a versatile in vitro reconstitution system that faithfully reproduces both branches of the type-V secretion pathway and the assembly of ß-barrel outer membrane proteins. This system will allow an in-depth analysis of protein secretion across and integration into outer membranes.

CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis.

Spore formation in Bacillus subtilis relies on a regulated intramembrane proteolysis (RIP) pathway that synchronizes mother-cell and forespore development. To address the molecular basis of this SpoIV transmembrane signaling, we carried out a structure-function analysis of the activating protease CtpB. Crystal structures reflecting distinct functional states show that CtpB constitutes a ring-like protein scaffold penetrated by two narrow tunnels. Access to the proteolytic sites sequestered within these tunnels is controlled by PDZ domains that rearrange upon substrate binding. Accordingly, CtpB resembles a minimal version of a self-compartmentalizing protease regulated by a unique allosteric mechanism. Moreover, biochemical analysis of the PDZ-gated channel combined with sporulation assays reveal that activation of the SpoIV RIP pathway is induced by the concerted activity of CtpB and a second signaling protease, SpoIVB. This proteolytic mechanism is of broad relevance for cell-cell communication, illustrating how distinct signaling pathways can be integrated into a single RIP module.

A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors.

The Ndc80 complex is the key microtubule-binding element of the kinetochore. In contrast to the well-characterized interaction of Ndc80-Nuf2 heads with microtubules, little is known about how the Spc24-25 heterodimer connects to centromeric chromatin. Here, we present molecular details of Spc24-25 in complex with the histone-fold protein Cnn1/CENP-T illustrating how this connection ultimately links microtubules to chromosomes. The conserved Ndc80 receptor motif of Cnn1 is bound as an α helix in a hydrophobic cleft at the interface between Spc24 and Spc25. Point mutations that disrupt the Ndc80-Cnn1 interaction also abrogate binding to the Mtw1 complex and are lethal in yeast. We identify a Cnn1-related motif in the Dsn1 subunit of the Mtw1 complex, necessary for Ndc80 binding and essential for yeast growth. Replacing this region with the Cnn1 peptide restores viability demonstrating functionality of the Ndc80-binding module in different molecular contexts. Finally, phosphorylation of the Cnn1 N-terminus coordinates the binding of the two competing Ndc80 interaction partners. Together, our data provide structural insights into the modular binding mechanism of the Ndc80 complex to its centromere recruiters.

Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in Arabidopsis.

Diverse stresses such as high salt conditions cause an increase in reactive oxygen species (ROS), necessitating a redox stress response. However, little is known about the signaling pathways that regulate the antioxidant system to counteract oxidative stress. Here, we show that a Glycogen Synthase Kinase3 from Arabidopsis thaliana (ASKα) regulates stress tolerance by activating Glc-6-phosphate dehydrogenase (G6PD), which is essential for maintaining the cellular redox balance. Loss of stress-activated ASKα leads to reduced G6PD activity, elevated levels of ROS, and enhanced sensitivity to salt stress. Conversely, plants overexpressing ASKα have increased G6PD activity and low levels of ROS in response to stress and are more tolerant to salt stress. ASKα stimulates the activity of a specific cytosolic G6PD isoform by phosphorylating the evolutionarily conserved Thr-467, which is implicated in cosubstrate binding. Our results reveal a novel mechanism of G6PD adaptive regulation that is critical for the cellular stress response.

Augmenting ß-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins.

ß-Barrel proteins are frequently found in the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. In Escherichia coli, these proteins are inserted in the outer membrane by the Bam (ß-barrel assembly machinery) complex, a multiprotein machinery formed by the ß-barrel protein BamA and the four peripheral membrane proteins BamB, BamC, BamD and BamE. The periplasmic part of BamA binds prefolded ß-barrel proteins by a ß-augmentation mechanism, thereby stabilizing the precursors prior to their membrane insertion. However, the role of the associated proteins within the Bam complex remains unknown. Here, we describe the crystal structure of BamB, a nonessential component of the Bam complex. The structure shows a typical eight-bladed ß-propeller fold. Two sequence stretches of BamB were previously identified to be important for interaction with BamA. In our structure, both motifs are located in close proximity to each other and contribute to a conserved region forming a narrow groove on the top of the propeller. Moreover, crystal contacts reveal two interaction modes of how BamB might bind unfolded ß-barrel proteins. In the crystal lattice, BamB binds to exposed ß-strands by ß-augmentation, whereas peptide stretches rich in aromatic residues can be accommodated in hydrophobic pockets located at the bottom of the propeller. Thus, BamB could simultaneously bind to BamA and prefolded ß-barrel proteins, thereby enhancing the folding and membrane insertion capability of the Bam complex.

The structure of the Myo4p globular tail and its function in ASH1 mRNA localization.

Type V myosin (MyoV)-dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2-tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail-lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.

Molecular transformers in the cell: lessons learned from the DegP protease-chaperone.

Structure-function analysis of DegP revealed a novel mechanism for protease and chaperone regulation. Binding of unfolded proteins induces the oligomer reassembly from the resting hexamer (DegP6) into the functional protease-chaperone DegP12/24. The newly formed cage exhibits the characteristics of a proteolytic folding chamber, shredding those proteins that are severely misfolded while stabilizing and protecting proteins present in their native state. Isolation of native DegP complexes with folded outer membrane proteins (OMPs) highlights the importance of DegP in OMP biogenesis. The encapsulated OMP beta-barrel is significantly stabilized in the hydrophobic chamber of DegP12/24 and thus DegP seems to employ a reciprocal mechanism to those chaperones assisting the folding of water soluble proteins via polar interactions. In addition, we discuss in this review similarities to other complex proteolytic machines that, like DegP, are under control of a substrate-induced or stress-induced oligomer conversion.

Formation of She2p tetramers is required for mRNA binding, mRNP assembly, and localization.

In eukaryotic cells, dozens to hundreds of different mRNAs are localized by specialized motor-dependent transport complexes. One of the best-studied examples for directional mRNA transport is the localization of ASH1 mRNA in Saccharomyces cerevisiae. For transport, ASH1 mRNA is bound by the unusual RNA-binding protein She2p. Although previous results indicated that She2p forms dimers required for RNA binding and transcript localization, it remained unclear if the dimer constitutes the minimal RNA-binding unit assembling in vivo. By using analytical ultracentrifugation we found that She2p forms larger oligomeric complexes in solution. We also identified a point mutant that shows impaired oligomer formation. Size-exclusion chromatography suggests that She2p forms defined tetramers at physiological concentrations. Subsequent structural studies by small-angle X-ray scattering confirmed this finding and demonstrated that the previously observed She2p dimers interact in a head-to-head conformation to form an elongated tetrameric complex. This She2p tetramer suggests the generation of large continuous RNA-binding surfaces at both sides of the complex. Biochemical studies and immunostaining of cells confirmed that She2p tetramer formation is required for RNA binding, efficient mRNP assembly, and mRNA localization in vivo. Our finding on She2p tetramerization resolves previously raised questions on complex formation and mRNP function.

Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes.

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.