ATG8-Interacting Motif: Evolution and Function in Selective Autophagy of Targeting Biological Processes

Autophagy is an evolutionarily conserved vacuolar process functioning in the degradation of cellular components for reuse. In plants, autophagy is generally activated upon stress and its regulation is executed by numbers of AuTophaGy-related genes (ATGs), of which the ATG8 plays a dual role in both biogenesis of autophagosomes and recruitment of ATG8-interacting motif (AIM) anchored selective autophagy receptors (SARs). Such motif is either termed as AIM or ubiquitin-interacting motif (UIM), corresponding to the LC3-interacting region (LIR)/AIM docking site (LDS) or the UIM docking site (UDS) of ATG8, respectively. To date, dozens of AIM or UIM containing SARs have been characterized. However, the knowledge of these motifs is still obscured. In this review, we intend to summarize the current understanding of SAR proteins and discuss the conservation and diversification of the AIMs/UIMs, expectantly providing new insights into the evolution of them in various biological processes in plants.

Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site

Kinases are important cancer biomarkers and are conventionally detected based on their catalytic activity. Kinases regulate cellular activities by phosphorylation of motif-specific multiple substrate proteins, resulting in a lack of selectivity of activity-based kinase biosensors. We present an alternative approach of sensing kinases based on the interactions of their allosteric docking sites with a specific partner protein. The new approach was demonstrated for the ERK2 kinase and its substrate ELK-1. A peptide derived from ELK-1 was bound to a gold electrode and ERK2 sensing was performed by electrochemical impedance spectroscopy. The sensors showed a high level of target selectivity for ERK2 when compared with p38gamma kinase and BSA. ERK2 was detected in its cellular concentration range, 0.2-8.0 microM. Using the flexibility of peptide design, our method is generic for developing sensitive and substrate-specific biosensors and other disease-related enzymes based on their interactions.

Distinct roles of haspin in stem cell division and male gametogenesis

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

STRESS FRACTURE AT DOCKING SITE AFTER BONE TRANSPORT MANAGED WITH INTRAMEDULLARY INTERLOCKING NAIL AND ILIAC AUTOGRAFT - A RARE CASE REPORT

INTRODUCTION : Distraction osteogenesis was introduced by Ilizarov and further developed by Cataneo et al. The concept of formation of new bone and soft tissues from distraction osteogenesis and freshening the sclerosed bone ends by corticotomy for stronger bony union and consolidation is applied here. Here we present a CASE REPORT : rare case of young adult operated by tibial bone transport using monolateral external xator, who presented to us after refracture near the docking site. We managed this patient with intramedullary reamed nailing and iliac crest autografting, showing good bony union and functional outcome at 3 months postoperatively. CONCLUSION : Thus from previous literature and our case ndings, we conclude that re-fractures and other complications of bone transport can be effectively managed by intramedullary reamed nailing leading to good bony union and fracture consolidation. The intramedullary nail provides rigid stability to both new bone regenerate due to callotasis and union at docking sit, signicantlydecreased rates of future refractures. This signicantly decreases patients morbidity and delivering satisfactory functional outcome.

The leukemic oncogene EVI1 hijacks a MYC super-enhancer by CTCF-facilitated loops

Chromosomal rearrangements are a frequent cause of oncogene deregulation in human malignancies. Overexpression of EVI1 is found in a subgroup of acute myeloid leukemia (AML) with 3q26 chromosomal rearrangements, which is often therapy resistant. In AMLs harboring a t(3;8)(q26;q24), we observed the translocation of a MYC super-enhancer (MYC SE) to the EVI1 locus. We generated an in vitro model mimicking a patient-based t(3;8)(q26;q24) using CRISPR-Cas9 technology and demonstrated hyperactivation of EVI1 by the hijacked MYC SE. This MYC SE contains multiple enhancer modules, of which only one recruits transcription factors active in early hematopoiesis. This enhancer module is critical for EVI1 overexpression as well as enhancer-promoter interaction. Multiple CTCF binding regions in the MYC SE facilitate this enhancer-promoter interaction, which also involves a CTCF binding site upstream of the EVI1 promoter. We hypothesize that this CTCF site acts as an enhancer-docking site in t(3;8) AML. Genomic analyses of other 3q26-rearranged AML patient cells point to a common mechanism by which EVI1 uses this docking site to hijack enhancers active in early hematopoiesis.

A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling

Stochastic signaling dynamics expand living cells’ information processing capabilities. An increasing number of studies report that regulators encode information in their pulsatile dynamics. The evolutionary mechanisms that lead to complex signaling dynamics remain uncharacterized, perhaps because key interactions of signaling proteins are encoded in intrinsically disordered regions (IDRs), whose evolution is difficult to analyze. Here we focused on the IDR that controls the stochastic pulsing dynamics of Crz1, a transcription factor in fungi downstream of the widely conserved calcium signaling pathway. We find that Crz1 IDRs from anciently diverged fungi can all respond transiently to calcium stress; however, only Crz1 IDRs from the Saccharomyces clade support pulsatility, encode extra information, and rescue fitness in competition assays, while the Crz1 IDRs from distantly related fungi do none of the three. On the other hand, we find that Crz1 pulsing is conserved in the distantly related fungi, consistent with the evolutionary model of stabilizing selection on the signaling phenotype. Further, we show that a calcineurin docking site in a specific part of the IDRs appears to be sufficient for pulsing and show evidence for a beneficial increase in the relative calcineurin affinity of this docking site. We propose that evolutionary flexibility of functionally divergent IDRs underlies the conservation of stochastic signaling by stabilizing selection.

Mechanistic dissection of dominant AIRE mutations in mouse models reveals AIRE autoregulation

The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.

Ctf3/CENP-I provides a docking site for the desumoylase Ulp2 at the kinetochore

The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle–regulated processes.

A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling

Stochastic signaling dynamics expand living cells’ information processing capabilities. An increasing number of studies report that regulators encode information in their pulsatile dynamics. The evolutionary mechanisms that lead to complex signaling dynamics remain uncharacterized, perhaps because key interactions of signaling proteins are encoded in intrinsically disordered regions (IDRs), whose evolution is difficult to analyze. Here we focused on the stochastic pulsing dynamics of Crz1, a transcription factor in fungi downstream of the widely conserved calcium signaling pathway. We find that Crz1 IDRs from anciently diverged fungi can all respond transiently to calcium stress; however, only Crz1 IDRs from the Saccharomyces clade support pulsatility, encode extra information, and rescue fitness, while the Crz1 IDRs from distantly related fungi do none of the three. On the other hand, we find that Crz1 pulsing is conserved in the distantly related fungi, consistent with the evolutionary model of stabilizing selection. Further, we show that a calcineurin docking site in a specific part of the IDRs appears to be sufficient for pulsing and show evidence for a beneficial increase in the relative calcineurin affinity of this docking site. We propose that evolutionary flexibility of functionally divergent IDRs underlies the conservation of stochastic signaling by stabilizing selection.