Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process. T he endosomal-lysosomal/vacuolar pathway is the primary catabolic system of eukaryotic cells that degrades extracellular and intracellular materials. Membrane proteins destined for degradation, such as misfolded proteins or endocytosed receptors, become tagged by ubiquitin for further sorting to the endosomal-lysosomal/vacuolar system for degradation (1). During this process, an evolutionarily conserved machinery called endosomal sorting complex required for transport (ESCRT), is responsible for sorting these ubiquitinated cargos into the intraluminal vesicles (ILVs) of prevacuolar compartments/multivesicular bodies (PVCs/MVBs), which subsequently fuse with vacuoles/lysosomes to deliver their contents into the lumen for proteolytic degradation (2, 3). Malfunction of the assembly or dissociation of the ESCRT machinery disrupts MVB formation and thus results in the accumulation of ubiquitinated membrane cargos (4, 5).Macroautophagy (hereafter as autophagy) is another highly conserved catabolic process, which converges on the endosomallysosomal/vacuolar pathway to deliver aberrant organelles, longlived proteins, and protein aggregates to the lysosome/vacuole via a unique structure termed the "autophagosome" (6). Morphologically different from MVBs, autophagosomes are characterized by a double membrane structure, which is initiated from the phagophore assembly site/preautophagosome site (PAS) (7). The proteins or organelles to be degraded are encapsulated by autophagosomes that fuse either directly with the vacuole/lysosome or with endosomes like MVBs for expansion/maturation to form amphisomes, which then fuse with vacuole/lysosome for degradation. A number of conserved autophagy-related gene (ATG) proteins have been identified as participating in the autophagy pathway in eukaryotic cells (8).Even though it is generally accepted that at least one population of...

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The endoplasmic reticulum is the cellular compartment in which secretory proteins are synthesized and folded. Perturbations of endoplasmic reticulum homeostasis lead to the accumulation of unfolded proteins. The activation of the unfolded protein response during endoplasmic reticulum stress transmits information about the status of protein folding to the cytosol and nucleus. The unfolded protein response leads to the upregulation of genes encoding endoplasmic reticulum chaperones, attenuation of translation, and initiation of the endoplasmic reticulum quality control system to restore endoplasmic reticulum homeostasis. When the unfolded protein response is insufficient to rebuild the steady state in endoplasmic reticulum, the programmed cell death or apoptosis would be initiated, by triggering cell injuries, even to cell death through apoptosis signals. In this review, we briefly outline research on the chaperones and foldases conserved in eukaryotes and plants, and describe the general principles and mechanisms of the endoplasmic reticulum quality control and the unfolded protein response. We describe the current models for the molecular mechanism of the unfolded protein response in plants, and emphasize the role of inositol requiring enzyme-1-dependent network in the unfolded protein response. Finally, we give a general overview of the directions for future research on the unfolded protein response in plants and its role in the response to environmental stresses.

Dormancy mechanisms in seeds and buds arrest growth until environmental conditions are optimal for development. A genotype-specific period of chilling is usually required to release dormancy, but the underlying molecular mechanisms are still not fully understood. To discover transcriptional pathways associated with dormancy release common to seed stratification and bud endodormancy, we explored the chilling-dependent expression of 11 genes involved in endoplasmic reticulum stress and the unfolded protein response signal pathways. We propose that endoplasmic reticulum stress and the unfolded protein response impact on seed as well as bud germination and development by chilling-dependent mechanisms. The emerging discovery of similarities between seed stratification and bud endodormancy status indicate that these two processes are probably regulated by common endoplasmic reticulum stress and unfolded protein response signalling pathways. Clarification of regulatory pathways common to both seed and bud dormancy may enhance understanding of the mechanisms underlying dormancy and breeding programs may benefit from earlier prediction of chilling requirements for uniform blooming of novel genotypes of deciduous fruit tree species.

It is widely recognized that the missing heritability of many human diseases is partially due to noncoding genetic variants, but there are multiple challenges that hinder the identification of functional diseaseassociated noncoding variants. The number of noncoding variants can be many times of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals, and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multigranular information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell-stage-specific regulatory roles three top novel regulatory elements on our list, respectively in the RET, RASGEF1A and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the affects the binding of the gliogenesis regulator .

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation ( Sox9 +/− ) with the Sox9 +/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9 +/Y440X mice survived, all Sox9 +/− mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9 +/Y440X compared with Sox9 +/− . Activating Sox9 Y440X specifically in the chondrocyte–osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9 +/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9 Y440X failed to interact with β-catenin and was unable to suppress transactivation of Ihh in cell-based assays . We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9 Y440X , cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

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Oncogenic mutations are associated with the activation of key pathways necessary for the initiation, progression and treatment-evasion of tumors. While large genomic studies provide the opportunity of identifying these mutations, the vast majority of variants have unclear functional roles presenting a challenge for the use of genomic studies in the clinical/therapeutic setting. Recent developments in predicting protein structures enable the systematic large-scale characterization of structures providing a link from genomic data to functional impact. Here, we observed that most oncogenic mutations tend to occur in protein regions that undergo conformation changes in the presence of the activating mutation or when interacting with a protein partner. By combining evolutionary information and protein structure prediction, we introduce the Evolutionary and Structure (ES) score, a computational approach that enables the systematic identification of hotspot somatic mutations in cancer. The predicted sites tend to occur in Short Linear Motifs and protein-protein interfaces. We test the use of ES-scores in genomic studies in pediatric leukemias that easily recapitulates the main mechanisms of resistance to targeted and chemotherapy drugs. To experimentally test the functional role of the predictions, we performed saturated mutagenesis in NT5C2, a protein commonly mutated in relapsed pediatric lymphocytic leukemias. The approach was able to capture both commonly mutated sites and identify previously uncharacterized functionally relevant regions that are not frequently mutated in these cancers. This work shows that the characterization of protein structures provides a link between large genomic studies, with mostly variants of unknown significance, to functional systematic characterization, prioritizing variants of interest in the therapeutic setting and informing on their possible mechanisms of action.