Biology subjects

Lindberg, I.

Publications and source records attributed to Lindberg, I..

7 recordsLinked to original sources

bioRxiv2026

A domain-swapped proPC1/3 structure reveals a conformational checkpoint for ER export

Prohormone convertase 1/3 (PC1/3, encoded by PCSK1) is a serine protease expressed in neuroendocrine cells that is required to produce insulin, glucagon-like peptide-1, adrenocorticotrophic hormone, and other peptide hormones. PC1/3 is synthesized as the zymogen proPC1/3, which undergoes autocatalytic maturation in the endoplasmic reticulum (ER) before trafficking to secretory granules. However, how autocatalytic maturation licenses the ER exit of PC1/3 remains unknown. Here, we determined the structure of an immature, catalytically inactive human proPC1/3S382A, which exits the ER as a domain-swapped homodimer. This domain-swapped conformation allows proPC1/3S382A to complete a conserved calcium pocket normally formed after autocatalysis and thereby become competent for ER exit. By defining this calcium pocket as a conformational checkpoint for ER export, our findings provide insights into the maturation of PC1/3 which may apply broadly to the PCSK family. Finally, our structure provides explanations for the functional consequences of many deleterious PCSK1 mutations.

biochemistry↗

bioRxiv2025

SEL1L-HRD1 ER-Associated Degradation Facilitates Prohormone Convertase 2 Maturation and Glucagon Production in Islet α Cells

Proteolytic cleavage of proglucagon by prohormone convertase 2 (PC2) is required for islet cells to generate glucagon. However, the regulatory mechanisms underlying this process remain largely unclear. Here, we report that SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD), a highly conserved protein quality control system responsible for clearing misfolded proteins from the ER, plays a key role in glucagon production by regulating turnover of the nascent proform of the PC2 enzyme (proPC2). Using a mouse model with SEL1L deletion in proglucagon-expressing cells, we observed a progressive decline in stimulated glucagon secretion and a reduction in pancreatic glucagon content. Mechanistically, we found that endogenous proPC2 is a substrate of SEL1L-HRD1 ERAD, and that degradation of misfolded proPC2 ensures the maturation of activation-competent proPC2 protein. These findings identify ERAD as a novel regulator of PC2 biology and an essential mechanism for maintaining cell function.

cell biology↗

bioRxiv2024

ProSAAS is Preferentially Secreted from Neurons During Homeostatic Scaling and Reduces Amyloid Plaque Size in the 5xFAD Mouse Hippocampus

The accumulation of {beta}-amyloid in Alzheimers disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha synuclein and {beta}-amyloid fibrillation in vitro, and is upregulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimers progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using Western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control (and/or reduced degradation). ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Interestingly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimers disease progression.

neuroscience↗

bioRxiv2022

SERCA2 regulates proinsulin processing and processing enzyme maturation in the pancreatic β cell

Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in both type 1 and type 2 diabetes; however, the mechanisms underlying impaired proinsulin processing remain incompletely understood. Here, we identify the sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) pump and {beta} cell ER Ca2+ as key regulators of systemic glucose tolerance and proinsulin processing. We generated mice with a {beta} cell-specific SERCA2 deletion ({beta}S2KO) and SERCA2 deficient INS-1 cells to show that SERCA2 loss increases systemic and pancreatic levels of proinsulin protein and leads to aberrant localization of proinsulin within the proximal {beta} cell secretory pathway. These defects in proinsulin processing were linked to reduced maturation of the proinsulin processing enzymes PC1/3 and PC2, suggesting a model whereby chronic ER Ca2+ depletion in the {beta} cell, which is observed in many pathological conditions, impairs the spatial regulation of prohormone trafficking, processing, and maturation within the {beta} cell secretory pathway.

physiology↗

bioRxiv2021

The proSAAS chaperone provides neuroprotection and attenuates transsynaptic α-synuclein spread in rodent models of Parkinson's disease

Parkinsons disease is a devastating motor disorder involving the aberrant aggregation of the synaptic protein synuclein (aSyn) and degeneration of the nigrostriatal dopaminergic tract. We previously showed that proSAAS, a small secreted chaperone protein widely expressed in neurons within the brain, is able to block aSyn-induced dopaminergic cytotoxicity in primary nigral neuron cultures. We show here that coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This positive functional outcome was accompanied by significant amelioration of the human aSyn-induced loss of both nigral tyrosine hydroxylase-positive cells and striatal tyrosine hydroxylase-positive terminals, demonstrating clear proSAAS-mediated protection of the nigro-striatal tract. ProSAAS overexpression also reduced the content of human aSyn protein in both the nigra and striatum and reduced the loss of tyrosine hydroxylase protein in both regions. Since proSAAS is a secreted protein, we tested the possibility that proSAAS is able to block the transsynaptic spread of aSyn from the periphery to the central nervous system, increasingly recognized as a potentially significant pathological mechanism. The number of human aSyn-positive neurites in the pons and caudal midbrain of mice following administration of human aSyn-encoding AAV into the vagus nerve was considerably reduced in mice coinjected with proSAAS-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. We suggest that proSAAS may represent a promising target for therapeutic development in Parkinsons disease. SignificanceThis paper describes two independent avenues of research that both provide support for the in vivo neuroprotective function of this small chaperone protein. In the first approach, we show that proSAAS overexpression provides remarkably effective protection against dopaminergic neurotoxicity in a rat model of Parkinsons disease. This conclusion is supported both by three independent assays of motor function as well as by quantitative analysis of surviving dopaminergic neurons in brain areas involved in the control of motor function. In the second line of research, we show that in mice, the spread of human synuclein across synapses can be blunted by proSAAS overexpression.

neuroscience↗

bioRxiv2020

A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils

Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.

neuroscience↗

bioRxiv2020

Phase separation of proSAAS into spheres results in core sequestration of TDP-43216-414 aggregates

As neurons age, protein homeostasis becomes less efficient, resulting in misfolding and aggregation. Chaperone proteins perform vital functions in the maintenance of cellular proteostasis, and chaperone-based therapies that promote sequestration of toxic aggregates may prove useful in blocking the development of neurodegenerative disease. We previously demonstrated that proSAAS, a small secreted neuronal protein, exhibits potent chaperone activity against protein aggregation in vitro, and blocks the cytotoxic effects of amyloid and alpha synuclein oligomers in cell culture models. We now examine whether cytoplasmic expression of proSAAS results in interaction with protein aggregates in this cellular compartment. We used site-directed mutagenesis, confocal microscopy, in vitro aggregation assays, and functional assays to investigate the interaction of proSAAS with TDP-43 and other known aggregating proteins. We report that expression of proSAAS within the cytoplasm generates dense, membrane-less 2 m proSAAS spheres which progressively fuse to form larger spheres, suggesting liquid droplet-like properties. ProSAAS spheres selectively accumulate a C-terminally truncated fluorescently-tagged form of TDP-43216-414, initiating its cellular redistribution by sequestration within the sphere core; these TDP-43216-414 -containing spheres also exhibit dynamic fusion. Removal of either the predicted -helix (37-70) composed of hydrophobic and charged amino acids or the stretch of amino acids encompassing the conserved hydrophobic region and the positively charged furin site (163-189) inhibits the ability of proSAAS both to form spheres and to encapsulate TDP-43 aggregates. As a functional output, we demonstrate that proSAAS expression results in cytoprotection against full-length TDP-43 toxicity in yeast. In summary, the normally secreted neuronal chaperone proSAAS, when expressed in the cytoplasm unexpectedly phase-separates to form spherical liquid-like condensates that undergo dynamic fusion. We conclude that cyto-proSAAS acts as a functional holdase for cytoplasmic TDP-43216-414 molecules via this phase-separation property, representing a cytoprotectant whose unusual biochemical properties can potentially be exploited in the design of therapeutic molecules.

neuroscience↗