Biology subjects

Silber, M.

Publications and source records attributed to Silber, M..

4 recordsLinked to original sources

bioRxiv2024

Glutamine availability regulates cDC subsets in tissue

AbstractProliferating tumor cells take up glutamine for anabolic processes engendering glutamine deficiency in the tumor microenvironment. How this might impact immune cells is not well understood. Using multiple mouse models of soft tissue sarcomas, glutamine antagonists, as well as genetic and pharmacological inhibition of glutamine utilization, we found that the number and frequency of conventional dendritic cells (cDC) is dependent on microenvironmental glutamine levels. cDCs comprise two distinct subsets - cDC1 and cDC2, with the former subset playing a critical role in antigen cross-presentation and tumor immunity. While both subsets show dependence on Glutamine, cDC1s are particularly sensitive. Notably, glutamine antagonism did not reduce the frequency of DC precursors but decreased proliferation and survival of cDC1s. Further studies suggest a role of the nutrient sensing mTOR signaling pathway in this process. Taken together, these findings uncover glutamine dependence of cDC1s that is coopted by tumors to escape immune responses. One Sentence SummaryType 1 conventional dendritic cells require glutamine to maintain their number in non-lymphoid tissue. SignificanceImmune evasion is a key hallmark of cancer; however, the underlying pathways are diverse, tumor-specific and not fully elucidated. Many tumor cells avidly import glutamine to support their anabolic needs, creating a glutamine-deficient tumor microenvironment (TME). Herein, using mouse models of soft tissue sarcomas, we show that glutamine depletion in TME leads to reduced type 1 conventional dendritic cells - a cell type that is critical for adaptive immune responses. This work is a paradigm for how tumor cell metabolism can regulate anti-tumor immune responses and will be foundational to future efforts targeting glutamine metabolism for cancer immunotherapy.

immunology↗

bioRxiv2021

Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state

The intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL- catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential- dependent oligomeric switch.

cell biology↗

bioRxiv2018

Stabilization / destabilization of the APP transmembrane domain by mutations in the di-glycine hinge alter helical structure and dynamics, and impair cleavage by γ-secretase

Intramembrane cleavage of the {beta}-amyloid precursor protein C99 substrate by {gamma}-secretase is implicated in Alzheimers disease pathogenesis. Since conformational flexibility of a di-glycine hinge in the C99 transmembrane domain (TMD) might be critical for {gamma}-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. CD, NMR and hydrogen/deuterium exchange measurements as well as MD simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the TMD. However, although helix destabilization/unfolding was not observed at the initial {varepsilon}-cleavage sites of C99, both mutants impaired {gamma}-secretase cleavage and altered its cleavage specificity. Moreover, helix flexibility enabled by the di-glycine hinge translated to motions of other helix parts. Our data suggest that both local helix stabilization and destabilization in the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can be conducive for productive substrate-enzyme interactions.

biophysics↗

bioRxiv2018

Increased γ-Site H-Bond Stability Relates to Altered ε-Efficiency and Aβ Levels in the I45T Familial Alzheimer’s Disease Mutant of APP

Cleavage of the amyloid precursor proteins (APP) transmembrane domain (TMD) by {gamma}-secretase is a crucial step in the aetiology of Alzheimers Disease (AD). Mutations in the APP TMD alter cleavage and lead to familial forms of AD (FAD). The majority of FAD mutations shift the preference of initial cleavage from {varepsilon}49 to {varepsilon}48, thus raising the AD-related A{beta}42/A{beta}40 ratio. The I45T mutation is among the few FAD mutations that do not alter {varepsilon}-site preference, while it dramatically reduces the efficiency of {varepsilon}-cleavage. Here, we investigate the impact of the I45T mutation on the backbone dynamics of the substrate TMD. Amide exchange experiments and molecular dynamics simulations in solvent and a lipid bilayer reveal an increased stability of amide hydrogen bonds at the {zeta}-and {gamma}-cleavage sites. Stiffening of the H-bond network is caused by an additional H-bond between the T45 side chain and the TMD backbone, which alters dynamics within the cleavage domain. In particular, the increased H-bond stability inhibits an upward movement of the {varepsilon}-sites in the I45T mutant. Thus, an altered presentation of {varepsilon}-sites to the active site of {gamma}-secretase as a consequence of restricted local flexibility provides a rationale for reduced {varepsilon}-cleavage efficiency of the I45T mutant.

biophysics↗