Biology subjects

Glickman, M. H.

Publications and source records attributed to Glickman, M. H..

7 recordsLinked to original sources

bioRxiv2025

Hypoxia-induced mitoROS triggers M1 linear ubiquitin chains and activates NF-κB signaling

Mitochondria are essential organelles responsible for cellular energy production and metabolism. Hypoxia, a pathophysiological condition, impairs the electron transport chain, disrupts mitochondrial function, and produces harmful reactive oxygen species (ROS). Ubiquitin signaling regulates mitochondrial health through several mechanisms, including protein degradation and mitophagy. Here, we show that hypoxia-induced mitophagy occurs independently of ubiquitination. However, mitochondria are heavily ubiquitinated under hypoxic stress. A significant portion of these hypoxia-induced ubiquitin chains constitute a specific type: linear head-to-tail fusions (M1), which are known for their role in NF-{kappa}B activation during cytokine signaling. We demonstrate that hypoxia-induced mitochondrial ROS leads to the accumulation of these M1 chains, activating NF-{kappa}B signaling and increasing the expression of its target genes. These findings reveal a critical internal signal that helps cells adapt to mitochondrial stress and triggers an inflammatory response.

biochemistry↗

bioRxiv2025

USP15: the fourth Proteasome-associated DUB

The human genome encodes approximately 100 deubiquitinating enzymes (DUBs), but only three are considered proteasome-associated DUBs (pDUBs): PSMD14/Rpn11, USP14, and UCHL5. Among these, only PSMD14 is an integral 19S subunit, whereas USP14 and UCHL5 bind transiently to specific proteasomal subunits. Given the dynamic nature of proteasome composition, we searched for additional pDUBs. USP15 was found to be associated with 26S proteasomes purified from cultured cells. In proteasome preparations from erythrocytes, USP15 was identified as the most abundant transient pDUB. It was even feasible to separate proteasomes containing USP15 from those containing USP14. Although USP15 utilizes an internal ubiquitin-like (UBL) domain for positioning itself at the proteasome, it did not compete with the UBL-containing USP14. USP15 facilitated substrate selection at the proteasome by efficiently disassembling short polyubiquitin (polyUb) chains, while sparing K48-linked tetra-ubiquitin conjugates from deubiquitination. This feature may aid the proteasome in differentiating between substrates to be rescued from those committed to proteolysis. Identification of a fourth pDUB encourages the continued search for additional proteasome-interacting proteins that modulate its substrate specificity in a context-specific manner.

biochemistry↗

bioRxiv2025

Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant, UBB+1

UBB{superscript 1}, a ubiquitin variant protein resulting from a frameshift in the ubiquitin-B gene, is a pathological hallmark of Alzheimer disease (AD). At the cellular level, UBB{superscript 1} disrupts the ubiquitin-proteasome system while inducing autophagy. Notably, UBB{superscript 1} itself is secreted via autophagosome-like vesicles. Here, we demonstrate that UBB{superscript 1} can be removed from the cell by degradative and secretory autophagy. SQSTM1/p62 functions as a pivotal ubiquitin receptor for UBB{superscript 1}, recognizing its ubiquitin domain and facilitating loading into autophagosomes. Oligomerization of SQSTM1/p62 was critical to isolate UBB{superscript 1} in bodies preventing its aggregation. Intriguingly, both gain- and loss-of-function SQSTM1/p62 suppressed UBB{superscript 1} secretion, causing intracellular retention: SQSTM1/p62 knockout led to UBB{superscript 1} accumulation in insoluble aggregates, while its overexpression promoted the formation of p62-UBB{superscript 1} bodies. We further identified distinct roles for SNARE-mediated membrane fusion in secretory autophagy of UBB{superscript 1}. Specifically, the R-SNARE SEC22B and the Q-SNAREs Syntaxin-4 (STX4) and SNAP23 participated in UBB{superscript 1} exocytosis. Disruption of SEC22B impaired the fusion of UBB{superscript 1}-containing autophagosomes with the plasma membrane, reducing UBB{superscript 1} secretion without affecting its intracellular turnover. Inhibition of lysosomes partially stabilized UBB{superscript 1} indicating that degradation and secretion are complementary processes that determine the fate of UBB+1. This study elucidates the dual roles of autophagy in managing neurotoxic proteins, highlighting SQSTM1/p62 as a key mediator of UBB{superscript 1} trafficking and secretion. Although ubiquitin typically acts as a degradation signal, our findings reveal a rare instance of a ubiquitin-related protein driving secretory autophagy. These findings advance our understanding of cellular mechanisms underlying the clearance of misfolded proteins in neurodegenerative diseases. SignificanceTo maintain health, cells must remove toxic proteins, typically by ubiquitin-dependent degradation. Neurons are particularly sensitive since they are not dividing and cannot replace damaged cells. This study suggests that autophagy, a pathway for degrading proteins, can also secrete harmful proteins, highlighting a new pathway for tackling neurodegenerative diseases. Specifically, UBB+1, a variant of the ubiquitin protein linked to Alzheimers disease, is recognized by a central autophagy component, p62, packaged into vesicles and secreted from cells. When p62 is absent, UBB+1 accumulates inside cells, which can be harmful. This work identifies UBB{superscript 1} as a novel cargo for secretory autophagy, extending the understanding of how cells may handle proteotoxic stress beyond classical degradation pathways.

cell biology↗

bioRxiv2023

A role for the proteasome alpha2 subunit N-tail in substrate processing

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the outer subunits. Free 20S CP is found in a latent state in which the N-termini of neighboring subunits form a gate blocking access to the channel. Entry of substrates can be facilitated by the attachment of the activators or regulatory particles, which can rearrange the blocking subunit N-terminal residues. In order to determine the specific physiological role of individual elements working in concert within the gate, we constructed a set of truncations or single-site mutations in each of the participating N-terminal tails. We report herein that whereas only a few N-termini are important for maintaining a closed gate, all seven N-termini participate in the open gate. Specifically, an invariant tyrosine (Y) in each subunit forms a hydrogen bond with a conserved aspartate (D) in the N-terminal tail of its counterclockwise neighbor, with the exception of the 1-2 pair leaving a gap in the ring circumference. A third residue (X) of this YD(X) motif aligns the open channel; specifically, the phenylalanine (F) at this position of the 2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of this 2 N-terminal tail slows down proteolysis despite the appearance of an open gate state. We conclude that the YD(X) motif in N-terminal tail of subunits plays an important role in gating the proteasome and in processing the substrate.

biochemistry↗

bioRxiv2022

ECPAS/Ecm29-Mediated 26S Proteasome Disassembly Is an Adaptive Response to Glucose Starvation

The 26S proteasome consists of loosely associated 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in eukaryotic cells exist as free 20S complexes; however, our mechanistic and physiological understanding of what determines the ratio of 26S to 20S species remains incomplete. Here, we show that glucose starvation in mammalian cells results in the uncoupling of 26S holoenzymes into intact 20S and 19S subcomplexes. Subcomplex affinity-purification and quantitative mass spectrometry revealed that Ecm29 proteasome adaptor and scaffold (ECPAS) is a crucial mediator of this structural remodeling. The loss of ECPAS abrogated 26S dissociation, leading to decreased degradation of 20S proteasome substrates such as puromycylated polypeptides and lysine-less cyclin B. In silico modeling analysis suggested that the conformational changes of ECPAS may commence the disassembly process. ECPAS was also essential for proper endoplasmic reticulum stress response and cell survival during glucose starvation. In addition, we evaluated the role of ECPAS in vivo using the mouse xenograft model and observed that glucose-deprived tumor tissues had significantly elevated 20S proteasome levels. Collectively, our results indicate that the 20S-19S disassembly mediated by ECPAS is a novel mechanism adapting global proteolysis to physiological needs and an effective cellular strategy against proteotoxic stress.

biochemistry↗

bioRxiv2020

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

Of all ubiquitin-like small protein modifiers, Rub1/NEDD8 is the closest kin of ubiquitin in sequence and in structure. Despite their profound similarities, prevalence of ubiquitin and of Rub1 is starkly different: targets of ubiquitin modification reach into the thousands, whereas unique targets of Rub1/NEDD8 appear limited to one family of proteins, Cullins. This distinction is likely due to dedicated E1 activating enzymes that select either one or the other and relay the modifier until it is covalently attached to a target. To convert typical neddylation targets for modification by ubiquitin, and vice versa, we designed reciprocal substitutions at position 72 of Rub1 and of ubiquitin to render them substrates for activation by their non-cognate E1 activating enzymes. We found that this single amino acid is sufficient to distinguish between Ub and Rub1 in living cells, and determine their targets. Thus, modification of Cullins by UbR72T could compensate for loss of Rub1, even as it maintained its ability to polymerize and direct conjugates for degradation. Conversely, Rub1T72R activated by ubiquitin-activating enzyme entered into the ubiquitination cascade, however was not efficiently polymerized, essentially capping polyubiquitin chains. Upon shortage of free ubiquitin under stress, even native Rub1 spilled-over into the ubiquitinome suppressing polyubiquitination. By contrast, the need to maintain monomeric modifications on unique targets is a likely explanation for why the Rub1-activating enzyme strictly discriminates against ubiquitin. Swapping Rub1 and ubiquitin signals uncovered a reason for maintaining two separate pathways across eukaryotic kingdom.

biochemistry↗

bioRxiv2019

Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex- the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.

biochemistry↗