Citations

Cytolytic immunosuppression with OKT3 antibody has been shown to be highly effective in reducing the frequency of early allograft rejection in cardiac transplantation. Failure of OKT3 immunosuppression in the absence of anti‐OKT3 antibodies is a rare event, having been reported in only 3 patients who manifested predominantly vascular (humoral) rejection during OKT3 therapy. We report the first case of hemodynamically serious cellular rejection, without a demonstrable vascular rejection component, in a cardiac allograft recipient during OKT3 treatment. This case underscores the need to remain vigilant for signs of rejection in cardiac recipients during OKT3 immunosuppression.

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.

Abstract Myeloma is a neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow. In these studies, we have demonstrated that CD40 is expressed in human myeloma cells and have used a recently established IL-6-dependent myeloma cell line, ANBL-6, to examine the potential function of CD40 expression in myeloma cells. In addition to its expression on the ANBL-6 cells, we show that CD40 is expressed on freshly isolated myeloma cells from seven of seven patients tested. To address the role of CD40 expression in myeloma cells, we have examined the responsiveness of the ANBL-6 cell line to a CD40-specific mAb, G28-5. This cell line has previously been shown to proliferate only in response to IL-6. Of interest in this study, G28-5 also induced proliferation of the ANBL-6 cells. This proliferation was substantially inhibited by an IL-6-neutralizing mAb. Analysis of ANBL-6 cell culture supernatants by ELISA demonstrated that G28-5-stimulated cells secreted significant levels of IL-6, whereas unstimulated cell culture supernatants contained undetectable levels of IL-6. Furthermore, CV-1/EBNA cells expressing the human CD40 ligand also induced the proliferation of the ANBL-6 cell line, an effect that was inhibited by the anti-IL-6 mAb. Lastly, RNA blot analysis demonstrated an increase in IL-6 message in G28-5-stimulated ANBL-6 cells over unstimulated cells. These results indicate that the primary mechanism of anti-CD40-stimulated proliferation of the ANBL-6 cells is the induction of autocrine IL-6 production. Moreover, these data suggest that the expression of CD40 in malignant plasma cells may play a role in tumor cell expansion, possibly by stimulating the induction of autocrine IL-6 secretion.

Abstract Hosts of plasma cell tumors have a depressed primary antibody response. We have investigated the ability of cell homogenates and culture fluids from short-term cultures of spleen cells and tumor cells of mice bearing the MOPC-315 plasmacytoma to suppress the in vivo primary antibody response to sheep red blood cells. The homogenates and culture fluids of both MOPC-315 spleen cells and tumor cells suppress the antibody response in a dose-dependent manner. Culture fluids of spleen cells from tumor-bearing mice contain a 10,000- to 20,000-dalton immunosuppressive factor. Culture fluids of non-adherent tumor cells contain a high m.w. suppressor. Injection of the high m.w. tumor suppressive factor into normal mice induces the expression or appearance of host cells that secrete the 10,000- to 20,000-dalton immunosuppressor. The tumor suppressive factor, but not the spleen factor, causes an alteration of lymphocyte membranes such that the anti-DNP activity of the MOPC-315 myeloma protein can be detected on the circulating lymphocytes of injected mice.

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.

Structural studies reveal that the V domain of HD-PTP has two tandem binding sites for binding ubiquitin. Like its yeast counterpart Bro1, HD-PTP binds the Vps4 MIT domain, stimulating its activity in a manner that is augmented by ubiquitin binding.

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.