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Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the C. elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved., Author summary As the most abundant protein in animals, collagen plays diverse roles and its dysregulation impacts aging and many fibrotic disorders. It is important to understand how premature collagen proteins in the ER are processed and secreted, as many other aspects of collagen regulation have been elucidated in mechanistic detail. In this paper, we have characterized a novel conserved family of TMEM39 proteins, including human TMEM39A and C. elegans tmem-39 that regulates ER stress response and collagen secretion. Human TMEM39A directly interacts with SEC23A, a core component of the COPII vesicle coating complex responsible for vesicular cargo secretion to the Golgi apparatus. The function of TMEM-39 proteins in collagen secretion appears highly conserved and independent to the ER stress response and the autophagy pathway. Our results provide insights into functions and mechanisms of TMEM39 proteins in collagen secretion and suggest it as a plausible target for tissue fibrotic diseases.

Circadian rhythms are generated by endogenous pacemakers that rely on transcriptional-translational feedback mechanisms conserved among species. In Drosophila, the stability of a key pacemaker protein PERIOD (PER) is tightly controlled by changes in phosphorylation status. A number of molecular players have been implicated in PER destabilization by promoting PER progressive phosphorylation. On the other hand, there have been few reports describing mechanisms that stabilize PER by delaying PER hyperphosphorylation. Here we report that the protein Suppressor of Ras (SUR-8) regulates circadian locomotor rhythms by stabilizing PER. Depletion of SUR-8 from circadian neurons lengthened the circadian period by about 2 hours and decreased PER abundance, whereas its overexpression led to arrhythmia and an increase in PER. Specifically SUR-8 promotes the stability of PER through phosphorylation regulation. Interestingly, downregulation of the protein phosphatase 1 catalytic subunit PP1-87B recapitulated the phenotypes of SUR-8 depletion. We found that SUR-8 facilitates interactions between PP1-87B and PER. Depletion of SUR-8 decreased the interaction of PER and PP1-87B, which supports the role of SUR-8 as a scaffold protein. Interestingly, the interaction between SUR-8 and PER is temporally regulated: SUR-8 has more binding to PER at night than morning. Thus, our results indicate that SUR-8 interacts with PP1-87B to control PER stability to regulate circadian rhythms., Author summary Circadian clocks govern daily rhythms in physiology and behavior. Conserved molecular machinery drives circadian clocks among animals. PERIOD is a key pacemaker protein in fruit flies that undergoes a series of post-translational modifications. Several kinases have been identified in destabilizing PER. Here we identify the role of SUR-8 in circadian locomotor rhythms. Depletion of SUR-8 in pacemaker neurons slows down circadian rhythms and reduces PER abundance. Indeed, SUR-8 promotes the stability of PER. Finally we characterize SUR-8 as a scaffold protein to bridge PER and a phosphatase (PP1-87B) together to regulate PER phosphorylation and abundance.

Breast cancer susceptibility gene 1 (BRCA1) and binding partner BRCA1-associated RING domain protein 1 (BARD1) form an essential E3 ubiquitin ligase important for DNA damage repair and homologous recombination. The Caenorhabditis elegans orthologs, BRC-1 and BRD-1, also function in DNA damage repair, homologous recombination, as well as in meiosis. Using functional GFP fusions we show that in mitotically-dividing germ cells BRC-1 and BRD-1 are nucleoplasmic with enrichment at foci that partially overlap with the recombinase RAD-51. Co-localization with RAD-51 is enhanced under replication stress. As cells enter meiosis, BRC-1-BRD-1 remains nucleoplasmic and in foci, and beginning in mid-pachytene the complex co-localizes with the synaptonemal complex. Following establishment of the single asymmetrically positioned crossover on each chromosome pair, BRC-1-BRD-1 concentrates to the short arm of the bivalent. Localization dependencies reveal that BRC-1 and BRD-1 are interdependent and the complex fails to properly localize in both meiotic recombination and chromosome synapsis mutants. Consistent with a role for BRC-1-BRD-1 in meiotic recombination in the context of the synaptonemal complex, inactivation of BRC-1 or BRD-1 enhances the embryonic lethality of mutants defective in chromosome synapsis. Our data suggest that under meiotic dysfunction, BRC-1-BRD-1 stabilizes the RAD-51 filament and alters the recombination landscape; these two functions can be genetically separated from BRC-1-BRD-1’s role in the DNA damage response. Together, we propose that BRC-1-BRD-1 serves a checkpoint function at the synaptonemal complex where it monitors and modulates meiotic recombination., Author summary Our genomes are passed down from one generation to the next through the specialized cell division program of meiosis. Meiosis is highly regulated to coordinate both the large scale chromosomal and fine scale DNA events to ensure fidelity. While the tumor suppressor BRCA1-BARD1 is essential for genome integrity, its specific role in meiosis has been difficult to uncover. Taking advantage of attributes of the Caenorhabditis elegans system, we have analyzed the function of the BRCA1-BARD1 complex in meiosis in this simple metazoan. We find that BRCA1 and BARD1 localize dynamically to the proteinaceous structure that aligns maternal and paternal chromosomes, where it regulates homologous recombination. Although BRCA1 and BARD1 mutants have only subtle meiotic defects, we show that this complex plays critical roles in meiotic recombination when meiosis is perturbed, and this is separable from BRCA1-BARD1’s function in response to DNA damage in somatic cells. These results highlight the complexity of ensuring accurate transmission of the genome and uncover the requirement for this conserved complex in meiosis.

Functional gastrointestinal disorders (FGIDs) are characterized by dysregulated gut-brain interactions. Emerging evidence shows that low-grade mucosal inflammation and immune activation contribute to FGIDs, including functional dyspepsia (FD). Stress plays an important role in the onset of FD symptoms. In human subjects with FD, presence of gastric mast cells has been reported, but factors that influence mast cell infiltration remain uncharacterized. Corticotropin-releasing factor (CRF) initiates the body's stress response and is known to degranulate mast cells. In this study, we delineated the role of the CRF system in the pathogenesis of FD in a rat model. Gastric irritation in neonate rat pups with iodoacetamide (IA) was used to induce FD-like symptoms. RNA interference (RNAi) was used to silence gastric CRF expression. Mast cell infiltrate in the stomach increased by 54% in IA-treated rats compared to controls and CRF-RNAi tended to decrease gastric mast cell infiltrate. Sucrose intake decreased in IA-treated rats and mast cell numbers showed a negative association with sucrose intake. IA treatment and transient silencing of gastric CRF increased hypothalamic CRF levels. In IA-treated rats, gastric levels of CRF receptor 2 (CRF2) decreased by ~76%, whereas hypothalamic CRF receptor 1 (CRF1) levels increased. Plasma levels of TNF-α showed a positive correlation with plasma CRF levels. Levels of phosphorylated p38 and ERK1/2 in the stomach showed a positive correlation with gastric CRF levels. Thus, CRF may contribute to low grade inflammation via modulating mast cell infiltration, cytokine levels, MAPK signaling, and the gut-brain axis.

In species ranging from humans to Caenorhabditis elegans, dietary restriction (DR) grants numerous benefits, including enhanced learning. The precise mechanisms by which DR engenders benefits on processes related to learning remain poorly understood. As a result, it is unclear whether the learning benefits of DR are due to myriad improvements in mechanisms that collectively confer improved cellular health and extension of organismal lifespan or due to specific neural mechanisms. Using an associative learning paradigm in C. elegans, we investigated the effects of DR as well as manipulations of insulin, mechanistic target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and autophagy pathways—processes implicated in longevity—on learning. Despite their effects on a vast number of molecular effectors, we found that the beneficial effects on learning elicited by each of these manipulations are fully dependent on depletion of kynurenic acid (KYNA), a neuroinhibitory metabolite. KYNA depletion then leads, in an N-methyl D-aspartate receptor (NMDAR)-dependent manner, to activation of a specific pair of interneurons with a critical role in learning. Thus, fluctuations in KYNA levels emerge as a previously unidentified molecular mechanism linking longevity and metabolic pathways to neural mechanisms of learning. Importantly, KYNA levels did not alter lifespan in any of the conditions tested. As such, the beneficial effects of DR on learning can be attributed to changes in a nutritionally sensitive metabolite with neuromodulatory activity rather than indirect or secondary consequences of improved health and extended longevity., Author summary Learning capacity is known to decline with age, and similar effects are also associated with several neurodegenerative diseases. Regulation of insulin signaling by dietary restriction (DR) modulates lifespan in many organisms, and it has been also shown to enhance learning and memory. However, the underlying mechanisms of these processes are largely unknown due to the difficulty in disentangling the systemic effects of DR from any potentially brain-specific effects. Here, we have analyzed the molecular effects of dietary restriction in C. elegans and show that associative learning is enhanced by reducing production of the tryptophan metabolite kynurenic acid (KYNA). KYNA is an antagonist of glutamatergic signaling in neurons, and we find that its depletion in the nervous system upon DR allows for increased activation of an interneuron that is both necessary and sufficient to mediate learning. Furthermore, we show that genetic or pharmacological modulation of diverse pathways known to be involved in the physiological responses to DR also enhance learning by reducing KYNA production, likely via the activation of a specific transcription factor. Finally, we demonstrate that KYNA levels have no effect on organismal lifespan, indicating that the effects of this KYNA-mediated response to dietary restriction is truly specific to brain function and not a secondary consequence of improved health or longevity. As altered KYNA levels are associated with neurodegenerative and psychiatric diseases, our results suggest that this component may be an important modulator of learning and memory in humans as well.

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites., Author Summary Certain parasitic flatworms pose serious threats to human health. Understanding more about the biology of flatworms can lead to new and better drug targets. However, due to the complex life cycles of parasitic flatworms, it has been suggested that a non-parasitic flatworm, Schmidtea mediterranea, be used to study flatworm biology. This worm can be used as a model to study processes that all flatworms have in common; these findings can be applied back to parasitic flatworms. Digestion occurs in all worms and relies on the activity of protease enzymes to break down protein. The authors were able to induce regurgitation in S. mediterranea and study the contents of the worm gut. These worms have several types of proteases present, although it seems that only two types, called cysteine and aspartyl proteases, are important in digestion in live worms. Furthermore, one specific cysteine protease, cathepsin B, is responsible for the bulk of the digestive capabilities in S. mediterranea. Cysteine proteases also appear to be involved in the ability of S. mediterranea to regenerate damaged body tissues. Understanding protease activity in S. mediterranea can provide insight as to the roles of these enzymes in medically relevant parasitic flatworms.