For more than 30 years, scientists have been trying to unravel the mystery of how a key biological molecule self assembles into a rogue protein-like substance known as amyloid, which is thought to play a role in the development of type 2 diabetes – a disease that affects 300 million people worldwide.
A team of scientists at the University of Leeds has, for the first time, been able to identify the step-by-step changes that take place in the molecule known as human islet amyloid polypeptide, or hIAPP, as it changes into amyloid.
They have also discovered new compounds that are able to speed up or slow down the process.
In healthy people, hIAPP is secreted by islets in the pancreas alongside the hormone insulin and it helps to regulate blood glucose levels and the amount of food in the stomach. When hIAPP malfunctions, it forms clumps of a protein-like substance called amyloid fibrils that kill the insulin-producing islets in the pancreas.
The build-up of amyloid fibrils is seen in people with type 2 diabetes although the exact mechanism of how it triggers disease is not known.
The research findings—Tuning the rate of aggregation of hIAPP into amyloid using small-molecule modulators of assembly—are published today in the journal Nature Communications.
The paper not only describes the complex molecular changes seen in hIAPP molecules as they transform into amyloid fibrils, but the scientists also announce that they have discovered two compounds, described as molecule modulators, which can control the process: one of the compounds delays it, the other accelerates it.
These molecule modulators can be used as “chemical tools” to help scientists investigate the way amyloid fibrils grow and how and why they become toxic.
Significantly they offer “starting points” for the development of drugs that could halt or control amyloid fibril formation and help in the urgent search to find ways to treat type 2 diabetes.
Sheena Radford, Royal Society Research Professor and Professor of Biophysics at the Astbury Centre for Structural Molecular Biology at Leeds, who supervised the research, said: “This is an exciting and huge step forward in our quest to understand and treat amyloid disease and to tackle a major health issue that is growing at an alarming rate.
“The compounds we have discovered are a first and important step towards small molecule intervention in a disease that has foxed scientists for generations.”
The research team looked at hIAPP found commonly in the population and a rare variant found in people with a genetic mutation known as S20G which puts them at greater risk of developing type 2 diabetes.
Amyloid fibril formation linked to disease
Understanding amyloid fibril formation is a key area of health research. The formation of fibrils is believed to be a factor in a range of life-limiting illnesses including Alzheimer’s Disease and Parkinson’s Disease, as well as type 2 diabetes.
Professor Radford added: “The results are also hugely exciting as they open the door to using the same type of approaches to understanding other amyloid diseases, the vast majority of which currently lack any treatments.”
Previous studies revealed that, under normal physiological conditions, transcriptional and posttranscriptional mechanisms drive IAPP synthesis in pancreatic β-cells ((58), (32), (59)). However, the relative impact and the importance of transcriptional mechanisms including promoter activity in IAPP turnover in stressed pancreatic β-cells are unclear and hence were investigated here.
The current study points to dynamic and transcriptionally-regulated IAPP turnover in stressed rat and human pancreatic β-cells, including the peptide’s accumulation in unexpected intracellular compartments, notably the nucleus. Notwithstanding, the synthesized hIAPP accumulated in ER/Golgi- and secretory vesicles-containing cytoplasmic fractions, presumably en route to the plasma membrane for storage and release.
High glucose- and thapsigargin-induced ER stress increased hIAPP accumulation in both nuclear and ER/Golgi fractions.
EM studies confirmed IAPP accumulation in the nucleus and other non-secretory organelles suggesting its export from biosynthetic compartments and/or reuptake. Thus, two distinct hIAPP trafficking pathways, a canonical (vesicular) en route to the plasma membrane and non-canonical (to cell nucleus), concurrently operate in β-cells under these disparate cellular conditions.
Previous studies demonstrated that plasma membrane cholesterol and non-vesicular lipids may bind to monomeric hIAPP to regulate its transport across the plasma membrane and other endogenous membranes, thus providing an alternative transporting platform for peptide trafficking in cells (60, 61).
Further insight into the intracellular localization of hIAPP came from the high-resolution confocal microscopy studies which revealed hIAPP accumulation in DAPI-deficient nucleolar regions (62). Nucleoli are membrane-less nuclear domains in eukaryotic cells and the primary site for ribosomal RNA biogenesis (50, 51).
The mammalian nucleolus has a classical tripartite structure consisting of the central-fibrillar component, middle-dense fibrillar component, and outer-granular component, each participating in specific stages of ribosomal RNA biogenesis (50, 51). In addition to the regulation of ribosomal RNA biosynthesis, (63), a recent study points to the important role of the nucleolus as a protein quality control center in cells (64). Nucleolin is a nucleolar phosphoprotein primarily localized in the dense fibrillar component of the nucleolus (65).
Accumulation of hIAPP in and around the center of nucleolin-positive areas in the human islet cells strongly indicates its association with a central fibrillar component of the nucleolus. Enhanced hIAPP accumulation within the nucleolin/TIA1-positive regions of human islet β-cells evoked by ER stress or high glucose conditions suggests a possible signaling and/or regulatory role for nucleolar hIAPP in the human islet cells under these adverse conditions.
Alternatively, β-cells may direct monomeric hIAPP to nucleolus for storage and/or refolding, thus adopting a recycling pathway of many other misfolded nuclear and cytosolic proteins (64). In some cells, the nucleolar aggresomes harbor aggregated proteins accumulating within or in close proximity of the nucleolus (66, 67). Nucleolar aggresomes usually consist of conjugated ubiquitin-labeled proteins and polyadenylated RNA (66).
Nucleolar aggresomes can be induced by various cellular stress conditions as well as compromised protein degradation (66). Because hIAPP has a strong propensity to aggregate, it is possible that nucleolar accumulation of hIAPP, at least in part, is due to sequestration and trafficking of (mis)folded but still soluble (non-aggregating) forms of hIAPP to the nucleolus, which may reduce its toxicity. Indeed, co-staining of T2DM human islets with hIAPP specific antibody and amyloid stain, Thioflavin-T (Th-T), revealed Th-T negative hIAPP assemblies in the nuclear regions of β-cells, thus making this scenario possible.
In line with this observation, cell fractionation studies revealed the accumulation of monomeric but not oligomeric hIAPP in the nucleus. Our biochemical studies further revealed that stress-induced hIAPP accumulation in the nucleus quantitatively accounts for a significant portion (~10–20%) of total hIAPP intracellular content. Interestingly, prolonged high glucose conditions or induction of ER stress co-induced expression of heat shock proteins, BIP and HSP70, as well as hIAPP in human islet β-cells, indicating their possible co-regulatory roles and function in stressed β-cells.
It has been shown that proinsulin C-peptide, a degradation fragment of proinsulin molecule, traffics to nucleoli to regulate the ribosomal rDNA transcription in cells (38). Recent studies reported that PGC1α, the master regulator of mitochondrial biogenesis, localizes to the nucleolus and regulates RNA polymerase-I transcription under various stress conditions in mouse models and humans (68). At present, the functional significance of the nucleolar localization of hIAPP is unclear.
It is well established that ribosomal RNA biogenesis is tightly coordinated with cell proliferation and growth, all of which are impacted during cellular stress (69). The previous study demonstrated that exogenous (extracellular) mouse IAPP stimulated mouse β-cell proliferation in a glucose-dependent manner by regulating Erk1 / 2 and Akt signaling cascade (70). Therefore, it is possible that nucleolar hIAPP also serves as a signaling molecule and regulates the biosynthesis of ribosomal subunits, which in turn affects the proliferative capability of β-cells.
However, only a minor fraction of proliferative β–cells harbored hIAPP in the nucleolus. Importantly, knockdown of hIAPP in human islets did not alter the expression of proliferation/cell cycle markers such as Ki67, evoked by high glucose. Similarly, hIAPP knockdown or overexpression had no significant effects on precursor or mature rRNA levels suggesting that hIAPP expression is dispensable for ribosomal biosynthesis. At least in this aspect, intracellular hIAPP differs from proliferative actions of extracellular proinsulin C-peptide and mouse IAPP (38, 70).
However, our study also revealed no negative impact of hIAPP nucleolar accumulation on β-cell proliferation or rRNA synthesis, suggesting that hIAPP accumulation in the nucleus may potentially serve a protective role against toxic hIAPP oligomers/aggregates that tend to accumulate in other cellular compartments such as cytoplasm or recycling organelles (Figs. 2–4).
On the other hand, intact nucleolar organization and preserved biosynthetic functions, notably RNA pol II activity, were required for hIAPP production preceding and during ER stress. In line with these findings, transient expression of FoxA2 dominant-negative (DN) form reduced hIAPP promoter activity in ER-stressed RINm5F β-cell line and primary human islet cells. Thus, activation of the FoxA2 signaling pathway, previously reported to regulate IAPP production under physiological conditions (31), is also required for hIAPP synthesis under stress conditions. In contrast to these findings, it has been reported that FoxA2 overexpression suppressed the expression of several β-cell genes including IAPP and insulin in the INS 832/13 cell line (71).
Different cellular backgrounds (INS vs. primary cells used in our study) and/or transfection strategies (stable vs. transient expression of DN FoxA2 constructs in our study) may lead to a different experimental outcome in the aforementioned FoxA2 functional studies. Importantly, our data are in line with a study by Shalev and colleagues showing a positive role for FoxA2 in IAPP expression in islet β-cells (31).
Interestingly, while each of HG, TG, and TN induced a significant (between 2 and 3.5 folds) increase in IAPP transcript levels respective to control human islets, they stimulated a minor and non-significant (1–1.4 folds) increase in steady-state mRNA levels of IAPP transcription factors, FoxA2 and PDX1 (Fig. 9).
In line with these transcriptomics results, we previously demonstrated that HG conditions also do not significantly change protein levels of FoxA2 (44). Collectively, these results suggest that HG and ER stress agents stimulate IAPP promoter activity and transcription, at least in part, by increasing the binding of FoxA2 and possibly PDX1 to IAPP promoter without altering their steady-state expression levels.
Therefore, promoter occupancy could serve as an important and limiting factor in IAPP transcription under various pathophysiological conditions, even in situations of constant expression levels of its two transcription factors. Although our study suggests that transcriptional factor FoxA2 and stress-responsive elements in hIAPP promoter play a major role in its synthesis during ER stress, we cannot rule out the possibility that ER-stress induced hIAPP synthesis is also regulated, at least in part, at the translational level.
Posttranscriptional mechanisms play an important role in hIAPP production under normal physiological conditions (58) and they may also play a regulatory role in hIAPP production in ER-stressed β-cells. In accord with this idea, potent transcriptional inhibitor, α-amanitin, despite completely disrupting nucleolar organization, only partially inhibited hIAPP synthesis in normal and ER-stressed human islets (Fig. S5).
An important question that still remains to be resolved is to which extent is a high glucose-stimulated hIAPP expression ER-stress dependent? The current and published studies clearly demonstrate that chronic HG treatment may concurrently induce ER stress and hIAPP transcription, thus supporting this scenario. Comparative transcriptomic studies (Fig. 9) revealed that HG treatment stimulated an increase in ER-stress marker BIP albeit at a reduced level compared to potent chemical ER stress inducers, TG, and TN.
On other hand, mRNA levels of a metabolic and ER stress marker, TXNIP, were markedly elevated in HG and to a lesser extent in TG/TN treated islets. Further, the hIAPP expression pattern in ER-stressed β-cells strongly correlated with TXNIP transcript levels. Relatedly, a recent study by Shalev and colleagues demonstrated the important regulatory role of TXNIP in FoxA2-mediated IAPP expression in normal and HG-treated islet β-cells (31).
Thus, our current study supports (or at least does not preclude) the idea that HG and ER-stress may stimulate hIAPP production via a shared TXNIP/FoxA2-signaling pathway. In line with this notion, CHIP studies showed that the binding of carbohydrate response element-binding protein (ChREBP) to the txnip promoter is augmented in HG and TG/TN treated β-cells, and is critically important for the enhanced TXNIP transcription under high glucose and ER-stress conditions (36, 57). However, the extent to which these TXNIP-mediated metabolic and stress pathways overlap and drive HG-induced hIAPP synthesis remains to be resolved in the future.
reference link :https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC8011634/
More information: Tuning the rate of aggregation of hIAPP into amyloid using small-molecule modulators of assembly, Nature Communications (2022). DOI: 10.1038/s41467-022-28660-7