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The success of vector management programs relies on knowledge of the biology and genetic make-up of mosquitoes so that they can be interlaced with modern tools for developing suitable intervention strategies. There are many reports available for rearing varied species of mosquito vectors. However, there are limited studies addressing the development of isofemale lines among mosquitoes to homogenize the population to obtain both high-quality genome assemblies and enrichment of phenotype. Anopheles stephensi, an urban malaria vector, is one of the major invasive vectors of malaria distributed throughout the Indian subcontinent, Middle East, and has recently been expanding its range in Africa. With the existence of three biological forms, distinctly identifiable based on the number of ridges on eggs with varying vectorial competence, An. stephensi is a perfect species for developing a method for the successful establishment of isofemale lines, which can be tested for retention of the expected vectorial competence for the various forms. We describe the key steps in the establishment and validation of isofemale lines, which include monitoring the transgenerational fitness traits, morphometrics of eggs, and adult wing size during every generation. After the initial inbreeding depression, as proof of the tedious selection process, no significant morphometric differences were observed in the wings and egg size between the parental and their respective isofemale lines. We observed a significant change in the vectorial competence between the respective isofemale and parental lines enriching expected differential susceptibility towards malaria parasites by the type and intermediate forms. Interestingly, IndCh and IndInt strains showed variations in resistance to different insecticides belonging to all the four major classes. These variant lines have been characterized for their levels of homozygosity both at the phenotype and genotype levels and can be used as a standard reference or as a biological resource for other studies related to urban malaria research.Author summaryIsofemale lines can be a valuable resource for characterizing and enhancing several genotypic and phenotypic traits. This is the first detailed report of the establishment of two isofemale lines of type and intermediate biological forms in Anopheles stephensi. The work encompasses the characterization of fitness traits among the two lines through a transgenerational study. Further, isofemale colonies were established and used to characterize insecticide susceptibility and vector competence. The study provides valuable insights into the differential susceptibility status of the parental and isofemale to different insecticides belonging to the same class. Corroborating with the earlier hypothesis we exemplify the high vector competence in type form than the intermediate from using homozygous isofemale lines. Using these isofemale lines it is now possible to study host-parasite interactions and identify factors that might be responsible for altered susceptibility and increased vector competence in An. stephensi biological forms that would also pave way for developing better vector management strategies.

Despite major advances in our understanding of players and mechanisms involved in peroxisome biogenesis and peroxisome degradation, very few studies have focused on unraveling the multi-layered connections between, and the coordination of, these two opposing processes that regulate peroxisome homeostasis. The intersection between these processes also provides exciting avenues for future research. This review highlights the links between peroxisome biogenesis and degradation, incorporating an integrative approach that is critical not only for a mechanistic understanding, but also for manipulating the balance between these processes in relevant disease models.

INTRODUCTION: Morphometric evaluation of the pedicle and isthmus of second cervical vertebra (C2) (Axis) is extremely vital before contemplating any surgical stabilization involving the Craniovertebral region, in view of its proximity to the vertebral artery and the cervical nerve root. The dimensions of pedicles and isthmuses in C2 vary between individuals and there is paucity of data in the Indian population. This study strives to measure the average pedicle and isthmus dimensions in a sample of population, which would enable selection of screws with safest diameters to be used in C2; thereby avoiding injury to adjacent neurovascular structures. MATERIALS AND METHODS: One Hundred patients in the age group between 18 and 70 years who underwent CT scan of head and neck region were included in the study. The aim of this study was to assess the anatomic suitability of transarticular and pedicle screw placement in Axis vertebrae of Indian population and determine the maximum safe diameter for screw placement. The following parameters were measured in millimeters: Pedicle width, Pedicle angle, Internal height and Isthmic height. RESULTS: The Mean maximum diameter of potential pedicle screw was 4.99 ± 1.1 mm for the right side with the left side being slightly wider at 5.20 ± 1.16 mm. Twenty eight (28%; 56 out of 200 pedicles) had a measurement

Anopheles stephensi is the most menacing malaria vector to watch for in newly urbanising parts of the world. Its fitness is reported to be a direct consequence of the vector adapting to laying eggs in over-head water tanks with street-side water puddles polluted by oil and sewage. Large frequent inversions in the genome of malaria vectors are implicated in adaptation. We report the genome assembly of a strain of An. stephensi of the type-form, collected from a construction site from Chennai (IndCh) in 2016. The genome reported here with a L50 of 4, completes the trilogy of high-resolution genomes of strains with respect to a 16.5 Mbp 2Rb genotype in An. stephensi known to be associated with adaptation to environmental heterogeneity. Unlike the reported genomes of two other strains, STE2 (2R+b/2Rb) and UCI (2Rb/2Rb), IndCh is found to be homozygous for the standard form (2R+b/2R+b). Comparative genome analysis revealed base-level details of the breakpoints and allowed extraction of 22,650 segregating SNPs for typing this inversion in populations. Whole genome sequencing of 82 individual mosquitoes from diverse geographical locations reveal that one third of both wild and laboratory populations maintain the heterozygous genotype of 2Rb. The large number of SNPs can be tailored to 1740 exonic SNPs enabling genotyping directly from transcriptome sequencing. The genome trilogy approach accelerated the study of fine structure and typing of an important inversion in An. stephensi, putting the genome resources for this understudied species on par with the extensively studied malaria vector, Anopheles gambiae. We argue that the IndCh genome is relevant for field translation work compared to those reported earlier by showing that individuals from diverse geographical locations cluster with IndCh, pointing to significant convergence resulting from travel and commerce between cities, perhaps, contributing to the survival of the fittest strain.

Macroautophagy (referred to hereafter as autophagy) is an intracellular degradation pathway in which the formation of a double-membrane vesicle called the autophagosome is a key event in the transport of multiple cytoplasmic cargo (e.g. proteins, protein aggregates, lipid droplets or organelles) to the vacuole (lysosome in mammals) for degradation and recycling. During this process, autophagosomes are formed de novo by membrane fusion events leading to phagophore formation initiated at the phagophore assembly site (PAS). In yeast, Atg11 and Atg17 function as protein scaffolds, essential for selective and non-selective types of autophagy, respectively. While Atg17 functions in non-selective autophagy are well-defined in the literature, less attention is concentrated on recent findings regarding the roles of Atg11 in selective autophagy. Here, we summarize current knowledge about the Atg11 scaffold protein and review recent findings in the context of its role in selective autophagy initiation and autophagosome formation.

Damaged or redundant peroxisomes and their luminal cargoes are removed by pexophagy, a selective autophagy pathway. In yeasts, pexophagy depends mostly on the pexophagy receptors, such as Atg30 for Pichia pastoris and Atg36 for Saccharomyces cerevisiae, the autophagy scaffold proteins, Atg11 and Atg17, and the core autophagy machinery. In P. pastoris, the receptors for peroxisomal matrix proteins containing peroxisomal targeting signals (PTSs) include the PTS1 receptor, Pex5, and the PTS2 receptor and co-receptor, Pex7 and Pex20, respectively. These shuttling receptors are predominantly cytosolic and only partially peroxisomal. It remains unresolved as to whether, when and how the cytosolic pools of peroxisomal receptors, as well as the peroxisomal matrix proteins, are degraded under pexophagy conditions. These cytosolic pools exist both in normal and mutant cells impaired in peroxisome biogenesis. We report here that Pex5 and Pex7, but not Pex20, are degraded by an Atg30-independent, selective autophagy pathway. To enter this selective autophagy pathway, Pex7 required its major PTS2 cargo, Pot1. Similarly, the degradation of Pex5 was inhibited in cells missing abundant PTS1 cargoes, such as alcohol oxidases and Fox2 (hydratase-dehydrogenase-epimerase). Furthermore, in cells deficient in PTS receptors, the cytosolic pools of peroxisomal matrix proteins, such as Pot1 and Fox2, were also removed by Atg30-independent, selective autophagy, under pexophagy conditions. In summary, the cytosolic pools of PTS receptors and their cargoes are degraded via a pexophagy-independent, selective autophagy pathway under pexophagy conditions. These autophagy pathways likely protect cells from futile enzymatic reactions that could potentially cause the accumulation of toxic cytosolic products. Abbreviations: ATG: autophagy related; Cvt: cytoplasm to vacuole targeting; Fox2: hydratase-dehydrogenase-epimerase; PAGE: polyacrylamide gel electrophoresis; Pot1: thiolase; PMP: peroxisomal membrane protein; Pgk1: 3-phosphoglycerate kinase; PTS: peroxisomal targeting signal; RADAR: receptor accumulation and degradation in the absence of recycling; RING: really interesting new gene; SDS: sodium dodecyl sulphate; TCA, trichloroacetic acid; Ub: ubiquitin; UPS: ubiquitin-proteasome system Vid: vacuole import and degradation

BackgroundAnopheles stephensi is the most menacing malaria vector to watch for in newly urbanizing parts of the world. The fitness is reported to be a direct consequence of the vector adapting to laying eggs in over-head water tanks with street-side water puddles polluted by oil and sewage. Large frequent inversions of malaria vectors are implicated in adaptation.ResultsWe report the assembly of a strain of An. stephensi of the type-form, collected from a construction site from Chennai (IndCh) in 2016. The genome completes the trilogy with respect to a 16 Mbp inversion (2Rb) in An. stephensi associated with adaptation to environmental heterogeneity. Comparative genome analysis revealed breakpoint structure and allowed extraction of 22,650 segregating SNPs for typing this inversion. Using whole genome sequencing of 82 individual mosquitoes, we conclude that one third of both wild and laboratory populations maintain heterozygous genotype of 2Rb. The large number of SNPs are tailored to assign inversion genotype directly from 1740 exonic SNPs 80% of which are expressed in various developmental stages.ConclusionsThe genome trilogy approach accelerates study of fine structure and typing of important inversions in malaria vectors putting the genome resources for the much understudied An. stephensi, on par with the extensively studied malaria vector, Anopheles gambiae. We argue that the IndCh genome is relevant for field translation work compared to those reported earlier by showing that individuals from diverse populations cluster with IndCh pointing to significant commerce between cities, perhaps, allowing for survival of the fittest strain.

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein–protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.

Background The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector’s genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution. Results To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role. Conclusion Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.

Closed traumatic dislocation of multiple metatarsophalangeal joints is a rare injury. Until now only one case of simultaneous dislocation of all five metatarsophalangeal joints has been reported in peer-reviewed studies. The complex anatomy of the metatarsophalangeal joints prevents the relocation of the joints in a closed manner in maximum cases. We are reporting a case of dorsal dislocation of the second to fifth metatarsophalangeal joints in the left foot after road traffic accident. Bony prominence over the plantar aspect and increased web space between toes on presentation, then incongruity of metatarsophalangeal joints has to be thoroughly checked on radiograph. Since closed reduction attempts failed open reduction was done through dorsal approach using two incisions. Button holing of the capsule with interposition of capsule and plantar plate was noted. Dorsal approach avoids damage to the plantar plate and surrounding soft tissues.

BackgroundThe mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector’s genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution.ResultsTo access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y-chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y-chromosome in any mosquito. By tracking full length mRNA for >15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively through male development and adulthood, suggesting an important role throughout male development.ConclusionCollectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate development of genetic control strategies of malaria transmission.

Late onset neurological deficit is a rare complication of spinal tuberculosis. Reactivation of the disease and compression by internal gibbus are the common causes for late onset neurological deficit. We report a rare cause of late onset paraplegia in a patient with post tubercular kyphotic deformity. The late onset neurological deficit was due to the adjacent segment degeneration proximal to the kyphotic deformity. Posterior hypertrophied ligamentum flavum and anterior disc osteophyte complex caused the cord compression. The increased stress for prolonged period at the end of the deformity was the reason for the accelerated degeneration. Patient underwent posterior decompression, posterolateral and interbody fusion. Deformity correction was not done. To our best knowledge, this is only the second report of this unusual cause of late onset paraplegia.

Background: Proximal Femoral Nail (PFN) provides tangible evidence for its usage in unstable pertrochanteric fractures. There are a veritable number of references which show that PFN by virtue of its intramedullary fixation reduces the tensile strain thereby mitigating implant failure. Ours is a prospective study done on 50 subjects to assess the outcome of PFN usage in unstable pertrochanteric fractures. Methodology: 50 patients with Jensen Michaelsen type III, IV, V intertrochanteric and reverse oblique fractures were included in the study. The reduction obtained intra operatively was assessed using the modified criteria of Baumgaertner. Follow up evaluations were done at 6, 12, 24 and 52 weeks thereafter. Clinical evaluation was done using the Mobility score of Parker and Palmer. Radiographic parameters like fracture union, screw slide and degree of varus collapse were also assessed. Statistical analysis was provided by Friedman test. Results: Jensen Michaelsen type IV was the most common pattern observed (44%). The mean pre-injury parker score was 8.4. Patients with reverse oblique fracture morphology, type V fractures and patients with osteoporosis were treated with Long PFN (19 cases). The mean operating time was 92 minutes (range 60 - 180), and the mean blood loss during the surgery was 145 ml (range 100ml - 320 ml). The reduction was good in 40 patients (80%) and acceptable in the rest. The average time taken for fracture union was 15 weeks. It was observed that patients reached their preoperative mobility score of Parker and Palmer by 6 months post-surgery which was statistically significant (χ2 = 217.642, p < .001). Discussion: Proximal Femoral Nailing is done through a minimally invasive approach not disturbing the fracture hematoma which is a vital in fracture consolidation. The biomechanics of intramedullary fixation in cases of destabilised medial cortex is optimised by medialization of the fulcrum point and resultant reduction of the bending moment with respect to proximal fixation. Fracture reduction was good in 80% cases and in 10 cases the reduction was acceptable with a mean varus malalignment of 13.2 degrees.The mean intraoperative blood loss of 145 ml in our series compares favourably with previously reported values in the literature. Most of the fractures in our series (66%) united by 12-14 weeks. The mean preoperative mobility score of Parker was 8.4, which was reached by patients on their 6th postoperative month (p & lt; 0.001). Each period showed statistically significant improvement over the previous period as evident from the rank total of the scores and the critical ratio.

Late onset paraplegia is a rare complication of spinal tuberculosis. Disease reactivation and cord compression by internal gibbus are the common causes for neurological deficit. We report a patient with post-tubercular kyphotic deformity in whom the late onset paraplegia is caused by ossified ligamentum flavum above the level of kyphotic deformity. The deficit was attributable to the ossified ligamentum flavum and she recovered completely following posterior decompression and instrumented posterolateral fusion. To the best of our knowledge, this is the first report of this unusual cause of late onset paraplegia.

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5S163R/wt), and homozygous knock‐in (Ctrp5S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.

TRIM37 gene mutations cause muscle–liver–brain–eye (mulibrey) nanism: a rare autosomal recessive, prenatal onset growth disorder. Wang et al. find that TRIM37 ubiquitylates and stabilizes PEX5, the receptor for import of peroxisomal matrix proteins, suggesting that mulibrey nanism is a new peroxisomal biogenesis disorder., Most proteins destined for the peroxisomal matrix depend on the peroxisomal targeting signals (PTSs), which require the PTS receptor PEX5, whose deficiency causes fatal human peroxisomal biogenesis disorders (PBDs). TRIM37 gene mutations cause muscle–liver–brain–eye (mulibrey) nanism. We found that TRIM37 localizes in peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with its C-terminal 51 amino acids (CT51), which is required for PTS protein import. PEX5 mutations (K464A or ΔCT51), or TRIM37 depletion or mutation, reduce PEX5 abundance by promoting its proteasomal degradation, thereby impairing its functions in cargo binding and PTS protein import in human cells. TRIM37 or PEX5 depletion induces apoptosis and enhances sensitivity to oxidative stress, underscoring the cellular requirement for functional peroxisomes. Therefore, TRIM37-mediated ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import, suggesting that mulibrey nanism is a new PBD.

One of the main unanswered questions regarding the early steps of macroautophagy/autophagy is the mechanism of membrane-modeling events required for autophagosome formation. Three independent studies have recently revealed an actin cytoskeleton involvement in this process, providing significant details regarding the role of actin in nucleation events both inside and outside the phagophore membrane during its expansion and assembly.

Purpose: Ensuring compliance to treatment protocol, especially regular visit to treating facility, is an important aspect of clubfoot management. However, the factors affecting compliance to follow-up schedule are myriad. Methods: A cross-sectional study was undertaken among caregivers of clubfoot patients from a tertiary referral clubfoot clinic in a developing country. Hospital records were reviewed to collect demographic data and subjects were classified as either “regular” or “irregular” if they missed ≤3 and >3 scheduled hospital visits, respectively. Various factors that could affect compliance such as family size, number of children, literacy of caregiver, occupation of breadwinner, and time taken to travel to hospital were studied. Caregivers were probed regarding the reason for their irregularity. Results: A total of 238 patients were included, of which 138 formed the “regular” group and the rest 100 formed the “irregular” group. Patients in the regular group were significantly younger (mean age 43.8 months) compared to the irregular group (59.8 months; p = 0.001). The mean follow-up period in the regular group was 28.1 months and in the irregular group was 33.8 months. On univariate analysis, age, duration of follow-up, and transport duration were found to be significant between the two groups. However, multivariate analysis revealed that female children with clubfoot are more likely to be irregular as compared to males ( p = 0.038). Conclusion: In a developing country setting, higher age and being a female child are associated with irregularity to hospital visit protocol. At clubfoot clinics, identifying these children and counseling their caregivers might improve compliance.

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.

Macroautophagy/autophagy, a catabolic process by which cytoplasmic materials are degraded and recycled in lysosomes/vacuoles, remains a rapidly expanding research topic with the need for constantly improved methodologies to study each step of this pathway. Recently Lee and colleagues, as well as Stolz et al., independently reported the development of new AIM/LIR-based fluorescent sensors, which mark individual endogenous mammalian Atg8-family (mAtg8) proteins without affecting the autophagic flux. When expressed in cells, each sensor selectively recognizes individual mAtg8 isoforms and distinguishes mammalian MAP1LC3/LC3 proteins from the related GABARAPs. Such selectivity was achieved by using various LC3-interacting regions with high binding affinity to either a subgroup, or a specific, mAtg8 isoform as part of the sensor. Here we discuss the utility of these sensors in autophagy research and highlight their strengths, weaknesses and future directions.

An intra-ER sorting process regulates segregation, packaging, and budding of peroxisomal importomer subcomplexes, thereby preventing their premature assembly at the ER., During de novo peroxisome biogenesis, importomer complex proteins sort via two preperoxisomal vesicles (ppVs). However, the sorting mechanisms segregating peroxisomal membrane proteins to the preperoxisomal endoplasmic reticulum (pER) and into ppVs are unknown. We report novel roles for Pex3 and Pex19 in intra–endoplasmic reticulum (ER) sorting and budding of the RING-domain peroxins (Pex2, Pex10, and Pex12). Pex19 bridged the interaction at the ER between Pex3 and RING-domain proteins, resulting in a ternary complex that was critical for the intra-ER sorting and subsequent budding of the RING-domain peroxins. Although the docking subcomplex proteins (Pex13, Pex14, and Pex17) also required Pex19 for budding from the ER, they sorted to the pER independently of Pex3 and Pex19 and were spatially segregated from the RING-domain proteins. We also discovered a unique role for Pex3 in sorting Pex10 and Pex12, but with the docking subcomplex. Our study describes an intra-ER sorting process that regulates segregation, packaging, and budding of peroxisomal importomer subcomplexes, thereby preventing their premature assembly at the ER.

Macroautophagy/autophagy is a highly conserved process in which subcellular components destined for degradation are sequestered within autophagosomes. The selectivity of autophagy is determined by autophagy receptors, such as Pichia pastoris Atg30 (autophagy-related 30), which controls the selective degradation of peroxisomes (pexophagy) through the assembly of a receptor-protein complex (RPC). Previously, we proved that the peroxisomal acyl-CoA-binding protein, Atg37, and the highly conserved peroxin, Pex3, are required for RPC formation and efficient pexophagy. Here, we describe how Atg37 and Pex3 regulate the assembly and activation of the pexophagic RPC. We demonstrate that Atg30 requires both Atg37 and Pex3 to recruit Atg8 and Atg11 to the pexophagic RPC, because Atg37 depends on Pex3 for its localization at the peroxisomal membrane. We establish that due to close proximity of Atg37- and Pex3-binding sites in the middle domain of Atg30, the binding of these proteins to Atg30 is mutually exclusive within this region. We also show that direct binding of Pex3 and Atg37 to Atg30 regulates its phosphorylation by the Hrr25 kinase, negatively and positively, respectively. Based on these results we present a model that clarifies the assembly and activation of the pexophagic RPC through the phosphoregulation of Atg30.

TRIM37 gene mutations cause mulibrey (muscle-liver-brain-eye) nanism, a severe growth disorder with prenatal onset. Although TRIM37 depletion normally induces apoptosis, patients with TRIM37 mutations have a high risk of developing tumors, suggesting that there may be an alternative pro-survival mechanism for TRIM37-deficient tumor cells. We find that TRIM37 interacts with MTOR and RRAGB proteins, enhances the MTOR-RRAGB interaction and promotes lysosomal localization of MTOR, thereby activating amino acid-stimulated MTORC1 signaling. In response to loss of TRIM37 functions, phosphorylation of TFEB is significantly reduced, resulting in its translocation into the nucleus enabling its transcriptional activation of genes involved in lysosome biogenesis and macroautophagy/autophagy. The enhanced autophagy depends on the inhibition of MTORC1 signaling and may serve as an alternative mechanism to survive the loss of TRIM37 functions. Our study unveils a positive role of TRIM37 in regulating the MTORC1–TFEB axis and provides mechanistic insights into the pathogenesis of mulibrey nanism, as well as potential therapeutic treatment. Abbreviations: ACTB: actin beta; ATG: autophagy related; CASP3: caspase3; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; CTS: cathepsin proteases; CTSL: cathepsin L; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LMNB1: lamin B1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mulibrey: muscle–liver–brain–eye; NAC: N-acetyl-L-cysteine; PARP1: poly(ADP-ribose) polymerase 1; RAP2A: member of RAS oncogene family; RHEB: Ras homolog enriched in brain; ROS: reactive oxygen species; RPS6KB1: ribosomal protein S6 kinase B1; RRAGB: Ras related GTP binding B; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TRIM37: tripartite motif containing 37.