Miroslav P. Milev

TRAMM/TrappC12 plays a role in chromosome congression, kinetochore stability, and CENP-E recruitment.

The TRAPP subunit TrappC12/TTC15, here renamed TRAMM, plays a role in the regulation of kinetochore stability and CENP-E recruitment during mitosis.

trappc11 is required for protein glycosylation in zebrafish and humans

The trappc11 mutation in zebrafish causes a stressed UPR, leading to fatty liver disease. This phenotype is not due to a trafficking defect but instead results from N-glycosylation defects. Hypoglycosylation and lipid accumulation are also found in patient cells with knockdown or mutated TRAPPC11 genes, suggesting a common etiology with zebrafish.

A novel TRAPPC11 mutation in two Turkish families associated with cerebral atrophy, global retardation, scoliosis, achalasia and alacrima

Background Triple A syndrome (MIM #231550) is associated with mutations in the AAAS gene. However, about 30% of patients with triple A syndrome symptoms but an unresolved diagnosis do not harbour mutations in AAAS. Objective Search for novel genetic defects in families with a triple A-like phenotype in whom AAAS mutations are not detected. Methods Genome-wide linkage analysis, whole-exome sequencing and functional analyses were used to discover and verify a novel genetic defect in two families with achalasia, alacrima, myopathy and further symptoms. Effect and pathogenicity of the mutation were verified by cell biological studies. Results We identified a homozygous splice mutation in TRAPPC11 (c.1893+3A>G, [NM_021942.5], g.4:184,607,904A>G [hg19]) in four patients from two unrelated families leading to incomplete exon skipping and reduction in full-length mRNA levels. TRAPPC11 encodes for trafficking protein particle complex subunit 11 (TRAPPC11), a protein of the transport protein particle (TRAPP) complex. Western blot analysis revealed a dramatic decrease in full-length TRAPPC11 protein levels and hypoglycosylation of LAMP1. Trafficking experiments in patient fibroblasts revealed a delayed arrival of marker proteins in the Golgi and a delay in their release from the Golgi to the plasma membrane. Mutations in TRAPPC11 have previously been described to cause limb-girdle muscular dystrophy type 2S (MIM #615356). Indeed, muscle histology of our patients also revealed mild dystrophic changes. Immunohistochemically, β-sarcoglycan was absent from focal patches. Conclusions The identified novel TRAPPC11 mutation represents an expansion of the myopathy phenotype described before and is characterised particularly by achalasia, alacrima, neurological and muscular phenotypes.

The TRAPP Subunit Trs130p Interacts with the GAP Gyp6p to Mediate Ypt6p Dynamics at the Late Golgi

Small GTPases of the Rab superfamily participate in virtually all vesicle-mediated trafficking events. Cycling between an active GTP-bound form and an inactive GDP-bound form is accomplished in conjunction with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Rab cascades have been described in which an effector of an activated Rab is a GEF for a downstream Rab, thus ensuring activation of a pathway in an ordered fashion. Much less is known concerning crosstalk between GEFs and GAPs although regulation between these factors could also contribute to the overall physiology of a cell. Here we demonstrate that a subunit of the TRAPP II multisubunit tethering factor, a Rab GEF, participates in the recruitment of Gyp6p, a GAP for the GTPase Ypt6p, to Golgi membranes. The extreme carboxy-terminal portion of the TRAPP II subunit Trs130p is required for the interaction between TRAPP II and Gyp6p. We further demonstrate that TRAPP II mutants, but not a TRAPP III mutant, display a defect in Gyp6p interaction. A consequence of this defective interaction is the enhanced localization of Ypt6p at late Golgi membranes. Although a ypt31/32 mutant also resulted in an enhanced localization of Gyp6p at the late Golgi, the effect was not as dramatic as that seen for TRAPP II mutants, nor was Ypt31/32 detected in the same TRAPP II purification that detected Gyp6p. We propose that the interaction between TRAPP II and Gyp6p represents a parallel mechanism in addition to that mediated by Ypt31/32 for the recruitment of a GAP to the appropriate membrane, and is a novel example of crosstalk between a Rab GAP and GEF.

TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of α-dystroglycan and muscular dystrophy

BackgroundTransport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation.Case presentationSubject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual.ConclusionRecessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy.

Mutations in TRAPPC12 Manifest in Progressive Childhood Encephalopathy and Golgi Dysfunction

Progressive childhood encephalopathy is an etiologically heterogeneous condition characterized by progressive central nervous system dysfunction in association with a broad range of morbidity and mortality. The causes of encephalopathy can be either non-genetic or genetic. Identifying the genetic causes and dissecting the underlying mechanisms are critical to understanding brain development and improving treatments. Here, we report that variants in TRAPPC12 result in progressive childhood encephalopathy. Three individuals from two unrelated families have either a homozygous deleterious variant (c.145delG [p.Glu49Argfs∗14]) or compound-heterozygous variants (c.360dupC [p.Glu121Argfs∗7] and c.1880C>T [p. Ala627Val]). The clinical phenotypes of the three individuals are strikingly similar: severe disability, microcephaly, hearing loss, spasticity, and characteristic brain imaging findings. Fibroblasts derived from all three individuals showed a fragmented Golgi that could be rescued by expression of wild-type TRAPPC12. Protein transport from the endoplasmic reticulum to and through the Golgi was delayed. TRAPPC12 is a member of the TRAPP protein complex, which functions in membrane trafficking. Variants in several other genes encoding members of the TRAPP complex have been associated with overlapping clinical presentations, indicating shared and distinct functions for each complex member. Detailed understanding of the TRAPP-opathies will illuminate the role of membrane protein transport in human disease.

TRAPPopathies: An emerging set of disorders linked to variations in the genes encoding transport protein particle (TRAPP)-associated proteins

The movement of proteins between cellular compartments requires the orchestrated actions of many factors including Rab family GTPases, Soluble NSF Attachment protein REceptors (SNAREs) and so‐called tethering factors. One such tethering factor is called TRAnsport Protein Particle (TRAPP), and in humans, TRAPP proteins are distributed into two related complexes called TRAPP II and III. Although thought to act as a single unit within the complex, in the past few years it has become evident that some TRAPP proteins function independently of the complex. Consistent with this, variations in the genes encoding these proteins result in a spectrum of human diseases with diverse, but partially overlapping, phenotypes. This contrasts with other tethering factors such as COG, where variations in the genes that encode its subunits all result in an identical phenotype. In this review, we present an up‐to‐date summary of all the known disease‐related variations of genes encoding TRAPP‐associated proteins and the disorders linked to these variations which we now call TRAPPopathies.

Bi-allelic mutations in TRAPPC2L result in a neurodevelopmental disorder and have an impact on RAB11 in fibroblasts

Background The combination of febrile illness-induced encephalopathy and rhabdomyolysis has thus far only been described in disorders that affect cellular energy status. In the absence of specific metabolic abnormalities, diagnosis can be challenging. Objective The objective of this study was to identify and characterise pathogenic variants in two individuals from unrelated families, both of whom presented clinically with a similar phenotype that included neurodevelopmental delay, febrile illness-induced encephalopathy and episodes of rhabdomyolysis, followed by developmental arrest, epilepsy and tetraplegia. Methods Whole exome sequencing was used to identify pathogenic variants in the two individuals. Biochemical and cell biological analyses were performed on fibroblasts from these individuals and a yeast two-hybrid analysis was used to assess protein-protein interactions. Results Probands shared a homozygous TRAPPC2L variant (c.109G>T) resulting in a p.Asp37Tyr missense variant. TRAPPC2L is a component of transport protein particle (TRAPP), a group of multisubunit complexes that function in membrane traffic and autophagy. Studies in patient fibroblasts as well as in a yeast system showed that the p.Asp37Tyr protein was present but not functional and resulted in specific membrane trafficking delays. The human missense mutation and the analogous mutation in the yeast homologue Tca17 ablated the interaction between TRAPPC2L and TRAPPC10/Trs130, a component of the TRAPP II complex. Since TRAPP II activates the GTPase RAB11, we examined the activation state of this protein and found increased levels of the active RAB, correlating with changes in its cellular morphology. Conclusions Our study implicates a RAB11 pathway in the aetiology of the TRAPPC2L disorder and has implications for other TRAPP-related disorders with similar phenotypes.

TRAMM, a new player in CENP-E biology

abstract Mitosis is a highly orchestrated process with morphologically defined stages and is subject to checkpoints that ensure the proper distribution of chromosomes. Centromere-associated protein E (CENP-E), a protein expressed during mitosis, is a potential target of cancer therapeutics. Our laboratory has recently implicated a protein called TRAMM (trafficking of membranes and mitosis) in the recruitment of CENP-E to kinetochores.