The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless. Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.

A general role for MIA3/TANGO1 in secretory pathway organization and function

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum, an essential process in eukaryotic cells. In vertebrates, the Mia3 gene encodes two major forms of Transport ANd Golgi Organization Protein 1 (TANGO1S and TANGO1L), previously implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics, and proteomics we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function including limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 and SURF4 to the ER-Golgi Intermediate Compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo including small soluble proteins. Our data support a general role for Mia3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells.

Myristoylation and its effects on the human Golgi Reassembly and Stacking Protein 55

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor and, when bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the protein's secondary structure, according to our circular dichroism data, it had a significant impact on the protein's thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

Continuous Glucose Monitoring in the Management of Hypoglycemia in TANGO2

Introduction: The TANGO2 gene encodes a transport and Golgi organization protein of unclear function; mutations should be considered in patients presenting with acute metabolic crisis, hypoglycemic episodes, cardiac arrhythmias, and other endocrinopathies. We report the novel use of a continuous glucose monitor (CGM) to help predict and prevent significant hypoglycemic episodes in a patient with TANGO2 mutation. Clinical Case: A 14-month old previously healthy, developmentally normal female who presented with unresponsive hypoglycemia (glucose 26 mg/dL) was demonstrated by Next Generation Sequencing to have a pathogenic 31.8 kb deletion of exon 3 to 9 in the TANGO-2 gene and a suspected pathogenic hemizygous c.569_592dup, p.Ile190_Leu197dup in TANGO-2. Her hospital course was notable for MRI showing hypoxic ischemic encephalopathy and both physical and electrical cardiac dysfunction. Continuous intravenous dextrose corrected the hypoglycemia, and transient hyperglycemia followed after several days of a glucose infusion rate between 3.2 to 5.8 mg/kg/min. After transitioning to ad lib oral feeds without restrictions, she was discharged. A second admission for acute unresponsive hypoglycemia and metabolic acidosis (glucose 30 mg/dL) occurred at 17 months of age with no clear inciting cause. Continuous IV dextrose at 9.9 mg/kg/min corrected the hypoglycemia and again resulted in transient hyperglycemia up to 271 mg/dl. Levothyroxine was also started for a TSH of 27 mIU/mL and a T4 of 4.6 ug/dL. Immediately after discharge, a DexCom G6 CGM was placed. Data over 2 weeks shows an average glucose of 104 ng/dL with 99% of the BS in target range. Parents report that CGM predictive low alerts have allowed intervention to abort fasting-related metabolic crises. Conclusion: In TANGO-2 deficiency, the liver may not adequately store and/or release glycogen in response to glucagon due to abnormal endoplasmic reticulum, Golgi apparatus, and mitochondrial functioning in states of stress or illness. Recent reports are conflicting with some showing reduced mitochondrial respiration in TANGO-2 patients in steady state with others finding normal values, opening the possibility that a combination of factors in the setting of stress may precipitate a metabolic crisis. Our patient quickly returns to near-normal physiological functioning; consequently, we suggest that use of a CGM can help prevent fasting related metabolic crisis in TANGO2 patients and can help guide feeding schedule and food choices to limit hyper- and hypoglycemia. In addition, CGM data can help further investigate if any beta cell dysregulation exists in non-acute states. References: Bérat CM, ... & de Lonlay P. (2020). Clinical and biological characterization of 20 patients with TANGO2 deficiency indicates novel triggers of metabolic crises.... J Inherit Metab Dis. 2020 Sep 14. doi: 10.1002/jimd.12314.

The SUN2-nesprin-2 LINC complex and KIF20A function in the Golgi dispersal

The morphology of the Golgi complex is influenced by the cellular context, which strictly correlates with nuclear functions; however, the mechanism underlying this association remains elusive. The inner nuclear membrane SUN proteins, SUN1 and SUN2, have diverse functions together with the outer nuclear membrane nesprin proteins, which comprise the LINC complex. We found that depletion of SUN1 leads to Golgi complex dispersion with maintenance of ministacks and retained function for vesicle transport through the Golgi complex. In addition, SUN2 associates with microtubule plus-end-directed motor KIF20A, possibly via nesprin-2. KIF20A plays a role in the Golgi dispersion in conjunction with the SUN2-nesprin-2 LINC complex in SUN1-depleted cells, suggesting that SUN1 suppresses the function of the SUN2-nesprin-2 LINC complex under a steady-state condition. Further, SUN1-knockout mice, which show impaired cerebellar development and cerebellar ataxia, presented altered Golgi morphology in Purkinje cells. These findings revealed a regulation of the Golgi organization by the LINC complex.

MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum. In vertebrates, this includes transport and Golgi organization protein 1 (TANGO1), encoded by the Mia3 gene. Here, using genome engineering of human cells light microscopy, secretion assays, and proteomics, we show loss of Mia3/TANGO1 results in formation of numerous vesicles and a loss of early secretory pathway integrity. This restricts secretion not only of large proteins like procollagens but of all types of secretory cargo. Our data shows that Mia3/TANGO1 constrains the propensity of COPII to form vesicles promoting instead the formation of the ER-Golgi intermediate compartment. Thus, Mia3/TANGO1 facilities the secretion of complex and high volume cargoes from vertebrate cells.

Tunnels for Protein Export from the Endoplasmic Reticulum

The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

SCYL1 arginine methylation by PRMT1 is essential for neurite outgrowth via Golgi morphogenesis

This study reveals that PRMT1 regulates the interaction between SCYL1 and γ2-COP for Golgi organization via SCYL1 arginine methylation. We propose that SCYL1 arginine methylation by PRMT1 contributes to axon and dendrite morphogenesis in neurons. This report contributes to the elucidation of brain development via arginine methylation.