Katzmann, David J.

ABSTRACT His domain protein tyrosine phosphatase (HD-PTP; also known as PTPN23) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, phosphoinositide 3-kinase (PI3K)/AKT and receptor tyrosine kinases (RTKs), such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. The ESCRT components Vps4 and Hrs have previously been implicated in cholesterol homeostasis; thus, these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.

AbstractThe Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT‐III polymers regulated by the AAA‐ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT‐associated Ub‐binding protein, to coordinate ESCRT‐III and Vps4‐dependent ILV biogenesis with upstream events such as cargo recognition.

HYLS1 regulates the ciliary phosphoinositide and hedgehog pathways, implicating a novel pathogenic mechanism for HLS syndrome.

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity but its mechanism of action was poorly understood. We report the high‐resolution crystal structures of the Did2‐ and Vps60‐binding N‐terminal domain and the Vps4‐binding C‐terminal domain of S. cerevisiae Vta1. The C‐terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C‐terminal domain stabilizes the ATP‐dependent double ring assembly of Vps4. In addition, the MIT motif containing N‐terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT‐III proteins. This provides an additional level of regulation and coordination for ESCRT‐III assembly and disassembly.

A key step in the lysosomal degradation of membrane proteins is sorting into the Multivesicular Body (MVB), a process wherein a region of the endosomal membrane buds into the lumen of the endosome to form an intraluminal vesicle (ILV). This sorting process is mediated by Endosomal Sorting Complexes Required for Transport (ESCRT‐0, I, II and III). Ubiquitination of MVB cargo and recognition of Ub‐cargo by ESCRT‐0, I and II permit entry into the MVB pathway; however, Ub is typically removed from the MVB cargo prior to cargo inclusion into the ILV. The coordination of deubiquination of MVB cargo with ESCRT‐III disassembly and ILV formation are not clear. Bro1 appears to play a critical role in coordinating cargo de‐ubiquitination and ILV formation through interactions with ESCRT machinery, the Ub peptidase, Doa4, ubiquitin‐modified cargo, and regulation of the ESCRT‐III‐associated AAA ATPase Vps4. These data suggest that members of the Bro1 family may be similarly impacting ESCRT‐dependent processes in a number of contexts. Grant Funding Source : NIH R01 GM07302

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

Human cholangiocytes, epithelial cells that line the bile ducts, were cultured as a model with which to study polarized small extracellular vesicle (sEV) communication. These cells release distinct sEV pools to mediate communication via their apical and basolateral domains. Furthermore, defective ESCRT function may contribute to disease states through altered sEV signaling.

ABSTRACT HIV protease is known to cause cell death, which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41, which directly binds Bak with nanomolar affinity, causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity, in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2, possibly explaining why those cells do not die when they reactivate HIV. Here, we determine that the Casp8p41-Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway, increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41-dependent manner. IMPORTANCE The Casp8p41 pathway of cell death is unique to HIV-infected cells yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV-infected cells to die.

Abstract TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors.