Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with wide distribution in the nervous system [1, 2]. It was first discovered in the hypothalamus, where it was found to activate cyclic adenosine monophosphate in anterior pituitary as well as it stimulates the release of pituitary hormones [3]. PACAP potently induces cyclic adenosine monophosphate (cAMP) levels in anterior pituitary cells as well as it stimulates the release of pituitary hormones. PACAP has two biologically active forms consisting of 38 amino acids (PACAP38) and 27 amino acids (PACAP27). The PACAP38 is the dominant form which undergoes internal cleavage-amidation resulting in the PACAP27 fragment [4,5,6]. PACAP is considered to be a member of the VIP/secretin/glucagon superfamily [4, 6, 7], and it shares structural similarities with vasoactive intestinal polypeptide (VIP) [8, 9]. The potent antagonist form of PACAP is residues 6–38 [10]. PACAP is synthesized as a precursor protein and is then cleaved to generate the active peptide. PreproPACAP is the precursor protein that contains a signal peptide. This signal peptide is cleaved to generate proPACAP, which is then further processed to produce the active peptide, PACAP.

PACAP binds to three G protein-coupled receptors, PAC1, VPAC1, and VPAC2. PAC1 is the most widely distributed of these receptors and plays an important role for mediating the effects of PACAP. The VPAC1 and VPAC2 receptors are also activated by VIP with similar affinity [11,12,13,14,15,16,17,18,19,20].

PACAP has a wide range of physiological actions. Among others, PACAP is involved in the regulation of pituitary hormone release [21, 22], the control of cardiovascular function [23], and the modulation of neuronal activity in the central and peripheral nervous systems [24, 25]. It has a well-known neurotrophic, neuroprotective and general cytoprotective effects based on its anti-inflammatory, antioxidant and anti-apoptotic properties. PACAP and its receptors are widely expressed in the nervous system, and they are involved in the regulation of neurogenesis, neuronal development and cell differentiation [26,27,28,29,30,31,32,33].

In previous investigations in cerebellar granule cells [34, 35], researchers reported contrasting effects of PACAP and ceramides on cellular migration, neurite outgrowth, and cytoskeletal remodeling, attributed to their opposing influences on actin distribution, tubulin polymerization, and Tau phosphorylation. To elaborate, C2-ceramide was associated with disrupted actin filament organization, tubulin depolarization, and altered microtubule-associated protein Tau, concomitant with impaired cell motility and neurite outgrowth. Conversely, PACAP exhibited a strengthening effect on actin polymerization at the growth cone, increased Tau phosphorylation, and counteracted the cytoskeletal perturbations induced by C2-ceramide. Furthermore, regarding actin and tubulin distribution and expression, PACAP treatment independently promoted actin polymerization at the growth cone, whereas it did not elicit remarkable alterations in tubulin cellular distribution. These previous findings highlight the distinct and opposing regulatory roles of PACAP and C2-ceramide in modulating critical aspects of cytoskeletal dynamics and cellular behavior.

Numerous in vivo animal models proved its neuroprotective effects. It has been discovered to protect neurons from oxidative stress, ischemia and different neuronal injuries [31,32,33]. Endogenous PACAP can also predict the outcome/mortality of patients who suffered traumatic brain injury [36, 37]. PACAP has been proposed as a therapeutic target for several neurological and psychiatric disorders, including Parkinson’s disease [38, 39], Alzheimer’s disease [39, 40], depression [41], and anxiety [42]. However, more research is needed to fully understand the therapeutic potential of PACAP and to develop effective PACAP-based therapies.

Aims of this study

The cytoskeleton, a dynamic network of structural proteins, plays a pivotal role in maintaining cellular shape, supporting intracellular transport, and facilitating cell movement. Among its key components, actin filaments stand out as major determinants of cytoskeletal architecture and function. Understanding the regulatory mechanisms governing actin dynamics is crucial for deciphering fundamental cellular processes.

While numerous studies have demonstrated the neuroprotective properties of PACAP, its role in tissue regeneration and potential as a therapeutic agent remains to be fully understood. Specifically, the understanding of PACAP’s impact on cytoskeletal dynamics, particularly the organization and disorganization of actin filament networks, is limited due to the scarcity of in vitro studies in this area. Additionally, the interaction between PACAP and actin has been minimally explored [34, 35], and the influence of PACAP on the thermal stability of actin is completely unknown. To build upon our understanding of PACAP's intricate interplay with the cytoskeleton, particularly actin dynamics, we employed differential scanning calorimetry (DSC) to investigate the thermodynamic aspects of PACAP–actin interactions.

In this context, our study focuses both on PACAP and its interactions with actin. Despite its potential therapeutic applications, the intricate interplay between PACAP and the cytoskeleton, especially actin, remains largely unexplored. Therefore, our research seeks to unravel the effects of PACAP on actin dynamics, shedding light on its significance in cytoskeletal regulation. Through a comprehensive investigation utilizing differential scanning calorimetry (DSC), we aim to elucidate the temperature-dependent alterations in Ca2+-G-actin stability induced by various forms and fragments of PACAP (see Fig. 1). Unveiling these interactions holds great promise for advancing our understanding of cytoskeletal biology and potentially paving the way for novel neuroprotective strategies.

Fig. 1
figure 1

Amino acid sequences of PACAP isoforms. PACAP 1-38 (P38, bioactive full-length natural isoform); PACAP 1-27 (P27, natural bioactive fragment); PACAP 6-27 (P6-27, artificial fragment); PACAP 6-38 (P6-38, artificial fragment)

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Materials and methods

Actin preparation

Ca2+-G-actin was prepared from rabbit striated muscle using a well-established protocol in our lab [43,44,45]. The MoPS buffer, essential for actin extraction and stabilization, was composed of MoPS(3-(N-morpholino) propane sulfonic acid) as the buffering agent, CaCl2 to maintain calcium ions at physiologically relevant levels, MEA (β-mercaptoethanol) as a reducing agent, and NaN3 to prevent bacterial contamination. Additionally, the buffer was supplemented with ATP (adenosine triphosphate) to maintain actin in its active state. The actin preparation also involved the inclusion of 50 mM KCl and 2 mM MgCl2 to support proper actin stability throughout the experiments. Rigorous care was taken to ensure the purity and integrity of the Ca2+-G-actin sample, thus guaranteeing the reliability and accuracy of subsequent investigations into the effects of PACAP on actin dynamics. We set the pH of the buffer at 7.8 which is essential to create a biologically relevant and controlled environment for studying actin dynamics and its interactions with PACAP. This approach ensures the stability of actin, increases the reliability of experimental results, and facilitates the understanding of how PACAP influences cytoskeletal regulation under conditions closely resembling those found in living systems. Purity of G-actin was verified by absorption photometry (clean peak at 280 nm) and by polyacrylamide gel electrophoresis (PAGE).

PACAP

For the applied PACAP forms and fragments and their amino acid sequences please check Fig. 1.

Differential Scanning Calorimetry (DSC) Measurements

The thermal unfolding experiments were performed by SETARAM Micro DSC-III in the 0–110 °C temperature range with 0.3 K min−1 scanning rate during two heating/cooling cycles. Conventional Hastelloy batch vessels were used during the denaturation experiments with 950 μL sample volume on average. (Conventional Hastelloy batch vessels are containers made of Hastelloy, a type of corrosion-resistant alloy composed mainly of nickel, chromium, and molybdenum. These vessels are commonly used in differential scanning calorimetry (DSC) experiments to hold the sample material and reference material during the measurement process. Hastelloy is chosen for its durability and resistance to corrosion, ensuring that the vessel does not react with the substances being analyzed, thereby preserving the integrity of the samples and providing accurate results in DSC experiments.) High-pressure pans and covers were utilized to maintain a sealed environment for the samples throughout the experiment, ensuring the integrity of the measurements and preventing any external influences on the samples during the differential scanning calorimetry (DSC) analysis.

The reference sample was the MoPS buffer. The sample and reference samples were equilibrated with a precision of ±0.1 mg. The buffer–buffer set was used as a baseline reference, which was subtracted from the original DSC curve. We have plotted the heat flow (DSC-III is a heat flux instrument with hermetically closed vessels) in the function of temperature. Calorimetric enthalpy was calculated from the area under the heat flow curve by using a two-point setting SETARAM peak integration. All results are given in mean values ± standard error (s.d., three measurements were performed in all cases).

For the assessment of thermal stability and interactions, Ca2+-G-actin was utilized at a consistent concentration of 2 mg mL−1 in all experiments. This concentration ensured the proper methodological conditions for DSC and allowed for meaningful comparisons across the experimental setup. As for the PACAP peptide, it was employed at a concentration of 21 µM, providing an optimal range for investigating potential interactions with actin under controlled experimental conditions. DSC measurements were performed with a highly sensitive calorimeter, enabling precise evaluation of the thermal denaturation and renaturation of Ca2+-G-actin in the presence of PACAP. The use of these standardized concentrations allowed for robust and reproducible data acquisition, facilitating a comprehensive understanding of PACAP's impact on actin stability and possible cytoskeletal regulation.

Results

In Fig. 2 and Table 1, the summarized result of the denaturation measurements of the physiologically important PACAP compounds can be seen in two heating–cooling cycles. The baseline reproducibility of our DSC is about ~ 2–3 µW, so we have to make more tests to check the reality of those scans that are lying close to this range. PACAP38 exhibited two endotherms at first heating around 40 and 57 °C and an exotherm at 91 °C. During the first cooling, we did not observe a detectable effect, which was surprising. However, in the second heating, the low-temperature denaturations were eliminated (indicating an irreversible change after the first heating), and the exotherm shifted to a higher value (~101 °C). The PACAP27 produced no endotherms but two exotherms in the first heating. During the second heating the first disappeared, but the second one remained at the same temperature with nearly the same ΔH. All effects were in the range of instrument error. These two derivatives should have a very stable core from a thermal point of view, which is supported by its interaction with G-actin (see Fig. 3 and Table 2).

Fig. 2
figure 2

Denaturation curves of PACAP38 and PACAP27 fragments. The curves are averages of two heating–cooling (h–c) cycles

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Table 1 Thermal parameters of different PACAP of 21 mM
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Fig. 3
figure 3

Denaturation curves of Ca2+-G actin and its complexes with different PACAP fragments during different heating cycles

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Table 2 Thermal parameters of 2 mg mL−1 (46 µM) Ca2+-G-actin (green) and its mixing with different PACAP of 21 µM
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In Fig. 3, the summarized result of the denaturation measurements can be seen performed on Ca2+-G actin mixed with PACAP compounds having physiologically relevant concentration. The Ca2+-G actin exhibited only a usual denaturation peak (Tm) around 56 °C with 0.023 Jg−1 calorimetric enthalpy (ΔHcal) [46].

The denaturation of Ca2+-G actin alone was irreversible, we did not detect a thermal signal either during the first cooling or during the second heating and cooling. In addition to Ca2+-G actin as the active form of PACAP [47], it shifted the transition temperature down to 55.7 °C, and at ~ 57 °C appeared a shoulder in the scan in case of first heating. This latter refers to the fading of the interaction between the two major domains of G-actin, which is a consequence of the binding of PACAP38 to the nucleotide-binding pocket of G-actin. We did not observe this strong effect for the other PACAP forms, which may emphasize a higher physiological importance of PACAP38. The whole denaturation temperature range decreased by ~ 4 °C, and at 98.7 °C appeared an exotherm with about half ΔHcal compared to the main denaturation (see Table 2). The cooling effect should be further investigated because it falls into the range of error of enthalpy detection of the instrument. The raw measurement data were smoothed by software, because a small effect is difficult to distinguish from the "base noise" of the instrument. These smoothed DSC curves show the tendency for a renaturation process. We need additional, more measurements so that averaging can reduce the role of noise, proving the fact of renaturation.

During the second heating, the actin signal disappeared (irreversibly denatured), and the exotherm exhibited lower Tm and at least a 40% smaller calorimetric enthalpy. This should be a PACAP signal, but its identification needs further investigation (see Fig. 2 and Table 1).

The PACAP27 fragment produced also an endotherm and exotherm. The endotherm’s Tm was a bit higher than in the case of PACAP38 with the same ΔHcal, but the exotherm exhibited 2.5 times higher calorimetric enthalpy during the first heating compared with PACAP38, while Tm remained practically the same. During the second heating, the endotherm was also absent, the exotherm peak decreased significantly producing closely the same ΔHcal as in the case of first heating (see Fig. 3 and Table 2).

The PACAP6-38 fragment exhibited nearly the same Tm but by 60% higher ΔHcal as was detected in the case of the Ca2+-G actin alone, without exotherm. The second heating cycle gave no detectable thermal effect because the DSC signal was in the range of instrument baseline fluctuation, that is the Ca2+-G actin PACAP6-38 complex underwent irreversible denaturation. This result is not shown in any figures or tables.

The first DSC scans of PACAP6-27 produced the same basic effect as was observed at PACAP6-38, but the Tm was the highest in our experiments with the smallest ΔHcal among the investigated complexes (see Fig. 3 and Table 2).

In line with prior in vitro studies [34, 35], our investigation has confirmed that PACAP exerts a substantial influence on the (thermal) stability of actin monomers, resulting in remarkable alterations in actin's structural and denaturation characteristics, potentially leading to functionally significant conformational changes. Nevertheless, a comprehensive exploration of these effects warrants further in-depth research.

Discussion

In this study, we conducted a comprehensive investigation on the effects of various PACAP fragments on the thermal stability of Ca2+-G actin using differential scanning calorimetry, and we also investigated the thermal stability of these PACAP forms and fragments alone (without Ca2+-G actin).

The DSC experiments conducted on physiologically relevant PACAP compounds in the absence of actin revealed intriguing thermal profiles. These findings suggest that both PACAP38 and PACAP27 possess a remarkably stable core (very likely that the red and green parts of the structure (see Fig. 1) are responsible for the exotherm process, while the missing red part is responsible for the absence of endotherm transition) from a thermal perspective, as evidenced by their resistance to denaturation and the observed interactions with G-actin. The presence of multiple thermal transitions implies complex structural changes or conformational rearrangements within these PACAP molecules. Further investigation is warranted to elucidate the precise nature and functional significance of these thermal transitions and their potential roles in biological processes because the PACAP molecules alone produced mild thermal effects in the investigated temperature range.

Regarding PACAP–actin complexes, our results demonstrated that Ca2+-G actin itself exhibits a typical denaturation peak, and the denaturation was irreversible, suggesting a significant structural alteration during this process. The addition of the active form of PACAP [47] to Ca2+-G actin induced a mild decrease (0.4 °C) in the denaturation temperature in the case of first heating, which could be a sign of loosening of the globular actin structure, because of the binding of PACAP into the cleft of actin. The exotherm effect at ~ 99 °C with relatively high ΔHcal (see Table 2) should refer to an aggregation of non-bound PACAP molecules. During the second heating, only the PACAP effect was detected with a significantly smaller peak temperature and ΔHcal, as in the case of a single PACAP sample. The first and second cooling produced an effect in the error range of the instrument, so we neglected it.

In the case of the PACAP27 fragment during the first heating cycle, the exotherm exhibited a significantly higher calorimetric enthalpy compared to PACAP38 (despite similar Tm values), which indicates more complex structural changes and greater ordering of the complex. This difference highlighted the unique thermodynamic behavior of PACAP27. In the case of the PACAP27-actin complex, this could imply that it undergoes more intricate or extensive conformational changes during heating compared to the PACAP38-actin complex. These changes may involve, initiating a larger number of bonds or interactions among PACAP molecules. This way higher ΔHcal can indicate greater overall stability of the complex. In other words, the complex might be held together by stronger forces or a higher number of interactions. This could mean that the PACAP27-actin complex has greater overall stability compared to the PACAP38-actin complex. During the second heating, the final stability of PACAP27 remained similar to the first heating result. The cooling phases should be also checked by performing more experiments or using different concentrations, because of the very low measured data.

Our results reveal that the PACAP6-38 and PACAP6-27 fragments, while exhibiting a denaturation temperature (Tm) similar to that of Ca2+-G actin alone, displayed a substantially higher calorimetric enthalpy, indicative of increased energy requirements for structural changes during denaturation. Importantly, the absence of an exothermic signal suggests a distinct denaturation process, highlighting the unique thermodynamic behavior of the PACAP6-38-actin and PACAP6-27-actin complexes.

In conclusion, it is the first attempt to investigate the actin-PACAP interaction with a thermoanalytical method. This way our study underscores the complex interplay between both natural PACAP forms [27, 47] and actin, revealing distinct thermodynamic profiles associated with different PACAP fragments. The notable shift observed when PACAP was added to Ca2+-G actin definitely indicates that the interaction between PACAP and actin alters the thermal stability of the actin. This shift can have important implications for understanding how PACAP influences the structure and stability of actin filaments, which are crucial components of the cytoskeleton and play essential roles in various cellular processes, including cell movement and shape maintenance. Our experimental findings obviously demonstrated that PACAP exerts a noticeable thermodynamic influence on the thermal stability and denaturation behavior of Ca2+-G actin. Notably, among the PACAP variants investigated, full-length PACAP (PACAP38), which is known for its substantial physiological regulatory activity alongside PACAP27, exhibited the most pronounced effect on actin thermal stability. Additionally, our study revealed that other examined PACAP forms and fragments (PACAP27, PACAP6-38, PACAP6-27) also induced alterations in the thermal stability profile of Ca2+-G actin, albeit to a lesser degree. These findings collectively underscore the various interactions between PACAP and actin, offering valuable insights into their thermodynamic behavior.

Our study also suggests that the complex formation between PACAP and actin is not merely an additive effect of their individual properties but involves specific interactions that modify the overall stability of the system. Further investigation is needed to precisely characterize these interactions and their functional consequences performing more experiments.

Conclusions

In conclusion, this study represents a pioneering effort to investigate the interaction between PACAP and G-actin using a thermoanalytical approach. The findings reveal distinct thermodynamic profiles associated with different PACAP fragments, emphasizing the complex interplay between PACAP forms [27, 47] and actin. The observed shift in thermal stability upon the addition of PACAP to Ca2+-G actin underscores the significant influence of PACAP on actin's structure and stability. Specifically, full-length PACAP (PACAP38) exhibits the most pronounced effect on actin thermal stability, highlighting its physiological regulatory activity alongside PACAP27. Additionally, other PACAP forms and fragments (PACAP27, PACAP6-38, PACAP6-27) induce alterations in the thermal stability profile of Ca2+-G actin, albeit to a lesser extent.

Furthermore, our results suggest that the complex formation between PACAP and G-actin involves specific interactions that modify the overall stability of the system, extending beyond a simple additive effect of their individual properties. The intricate thermodynamic behavior observed, including multiple thermal transitions and changes in calorimetric enthalpy, underscores the complexity of the PACAP-actin interaction and its potential implications for cellular processes such as cell movement and shape maintenance. However, further investigation (mainly PACAP interactions with F-actin) is warranted to precisely characterize these interactions and understand their functional consequences, necessitating additional experiments and analyses. Overall, this study provides valuable insights into the thermodynamic dynamics of the PACAP-actin interaction, laying the groundwork for future research in this field.