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Abstract Background Lupus nephritis (LN) is an inflammatory disease of the kidneys affecting patients with systemic lupus erythematosus. Current immunosuppressive and cytotoxic therapies are associated with serious side effects and fail to protect 20–40% of LN patients from end-stage renal disease. In this study, we investigated whether a small heat shock protein, HSPB5, can reduce kidney inflammation and the clinical manifestations of the disease in NZB/W F1 mice. Furthermore, we investigated whether HSPB5 can enhance the effects of methylprednisolone, a standard-of-care drug in LN, in an endotoxemia mouse model. Methods NZB/W F1 mice were treated with HSPB5, methylprednisolone, or vehicle from 23 to 38 weeks of age. Disease progression was evaluated by weekly proteinuria scores. At the end of the study, the blood, urine, spleens, and kidneys were collected for the assessment of proteinuria, blood urea nitrogen, kidney histology, serum IL-6 and anti-dsDNA levels, immune cell populations, and their phenotypes, as well as the transcript levels of proinflammatory chemokine/cytokines in the kidneys. HSPB5 was also evaluated in combination with methylprednisolone in a lipopolysaccharide-induced endotoxemia mouse model; serum IL-6 levels were measured at 24 h post-endotoxemia induction. Results HSPB5 significantly reduced terminal proteinuria and BUN and substantially improved kidney pathology. Similar trends, although to a lower extent, were observed with methylprednisolone treatment. Serum IL-6 levels and kidney expression of BAFF, IL-6, IFNγ, MCP-1 (CCL2), and KIM-1 were reduced, whereas nephrin expression was significantly preserved compared to vehicle-treated mice. Lastly, splenic Tregs and Bregs were significantly induced with HSPB5 treatment. HSPB5 in combination with methylprednisolone also significantly reduced serum IL-6 levels in endotoxemia mice. Conclusions HSPB5 treatment reduces kidney inflammation and injury, providing therapeutic benefits in NZB/W F1 mice. Given that HSPB5 enhances the anti-inflammatory effects of methylprednisolone, there is a strong interest to develop HSBP5 as a therapeutic for the treatment of LN.

Small heat shock proteins (SHSPs) are conserved proteins that participate in many cellular functions like preventing protein aggregation and stress response. However, their role in responding to nanoparticles (NPs) has not yet been explained. We used a chicken embryo model to investigate the effects of two different forms of iron oxide-NPs (IONPs) on the mRNA expression of HSPB1, HSPB5, HSPB8, and HSPB9 in cerebral tissue. Two hundred-ten fertilized eggs were randomly divided into seven groups (30 eggs/group; 10 eggs/replicate). Three groups received 100 ppm, 250 ppm, and 500 ppm of Fe2O3-NPs, respectively. Three other groups received 100 ppm, 250 ppm, and 500 ppm of Fe3O4-NPs, respectively, and one group remained untreated as a control. The NPs were given by in ovo method (0.3 ml/egg) only once on the first day of the embryonic period. Samples from cerebrums were collected on day 20 for gene expression analyses. HSPB1, HSPB5, HSPB8, and HSPB9 were all expressed in both normal and IONPs exposed cerebrums. SHSPs tested were differentially expressed in response to various concentrations of IONPs. The highest expression levels in response to Fe2O3-NPs and Fe3O4-NPs were observed for HSPB5 and HSPB9, respectively. The greatest gene expression changes due to the Fe2O3-NPs and Fe3O4-NPs exposure observed for HSPB1 and HSPB5, respectively. The results suggest a protective cellular mechanism against IONPs through SHSPs and recommend that expression profiling of SHSPs be included in the study of nanotoxicity. [ABSTRACT FROM AUTHOR]

[Display omitted] • α-crystallins (αAc and αBc) are archetypal sHSPs vital for proteostasis. • αAc and αBc display client-induced expansion and elongation of sHSP scaffold. • Client-saturation results in light-scattering amorphous sHSP co-aggregates. • Co-aggregates maintain internal features of highly elongated sHSP scaffolding. • The quasi-ordered trajectory reshapes our understanding of sHSP aggregation. Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined structural changes in αAc, αBc and native heteromeric lens α-crystallins (αLc) in their apo-states and at varying degree of chaperone saturation leading to co-aggregation, using lysozyme and insulin as model clients. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP oligomeric scaffold, whereby the native cage-like sHSP assembly displays a directional growth to accommodate saturating conditions of client sequestration. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP oligomers with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across αAc, αBc and αLc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of α-crystallins, carrying potential implications for a pathway toward cataract formation. [ABSTRACT FROM AUTHOR]

Background: HSPB5 is an ATP-independent molecular chaperone that is induced by heat shock or other proteotoxic stresses. HSPB5 is cytoprotective against stress both intracellularly and extracellularly. It acts as a potential therapeutic candidate in ischemia-reperfusion and neurodegenerative diseases. Results: In this paper, we constructed a recombinant plasmid that expresses and extracellularly secrets a HSPB5-Fc fusion protein (sHSPB5-Fc) at 0.42 μg/ml in CHO-K1 cells. This sHSPB5-Fc protein contains a Fc-tag at the C-terminal extension of HSPB5, facilitating protein-affinity purification. Our study shows that sHSPB5-Fc inhibits heat-induced aggregation of citrate synthase in a time and dose dependent manner in vitro. Administration of sHSPB5-Fc protects lens epithelial cells against cisplatin- or UVB-induced cell apoptosis. It also decreases GFP-Httex1-Q74 insolubility, and reduces the size and cytotoxicity of GFP-Httex1-Q74 aggregates in PC-12 cells. Conclusion: This recombinant sHSPB5-Fc exhibits chaperone activity to protect cells against proteotoxicity. [ABSTRACT FROM AUTHOR]

Sensorineural hearing loss is the most common long-term deficit after pneumococcal meningitis (PM), occurring in up to 30% of surviving patients. The infection and the following overshooting inflammatory host response damage the vulnerable sensory cells of the inner ear, resulting in loss of hair cells and spiral ganglion neurons, ultimately leading to elevated hearing thresholds. Here, we tested the oto-protective properties of the small heat shock protein alpha B-crystallin (HspB5) with previously reported anti-inflammatory, anti-apoptotic and neuroprotective functions, in an experimental model of PM-induced hearing loss. We analyzed the effect of local and systemic delivery of HspB5 in an infant rat model of PM, as well as ex vivo , using whole mount cultures. Cytokine secretion profile, hearing thresholds and inner ear damage were assessed at predefined stages of the disease up to 1 month after infection. PM was accompanied by elevated pro-inflammatory cytokine concentrations in the cerebrospinal fluid (CSF), leukocyte and neutrophil infiltration in the perilymphatic spaces of the cochlea with neutrophils extracellular trap formation during the acute phase of the disease. Elevated hearing thresholds were measured after recovery from meningitis. Intracisternal but not intraperitoneal administration of HspB5 significantly reduced the levels of TNF-α, IL-6 IFN-γ and IL-10 in the acute phase of the disease. This resulted in a greater outer hair cell survival, as well as improved hearing thresholds at later stages. These results suggest that high local concentrations of HspB5 are needed to prevent inner ear damage in acute PM. HspB5 represents a promising therapeutic option to improve the auditory outcome and counteract hearing loss after PM. [ABSTRACT FROM AUTHOR]

Summary: Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by demyelination, inflammation and neurodegeneration throughout the central nervous system. Although spinal cord pathology is an important factor contributing to disease progression, few studies have examined MS lesions in the spinal cord and how they differ from brain lesions. In this study we have compared brain and spinal cord white (WM) and grey (GM) matter from MS and control tissues, focusing on small heat shock proteins (HSPB) and HSP16.2. Western blotting was used to examine protein levels of HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 in brain and spinal cord from MS and age‐matched non‐neurological controls. Immunohistochemistry was used to examine expression of the HSPs in MS spinal cord lesions and controls. Expression levels were quantified using ImageJ. Western blotting revealed significantly higher levels of HSPB1, HSPB6 and HSPB8 in MS and control spinal cord compared to brain tissues. No differences in HSPB5 and HSP16.2 protein levels were observed, although HSPB5 protein levels were higher in brain WM versus GM. In MS spinal cord lesions, increased HSPB1 and HSPB5 expression was observed in astrocytes, and increased neuronal expression of HSP16.2 was observed in normal‐appearing GM and type 1 GM lesions. The high constitutive expression of several HSPBs in spinal cord and increased expression of HSPBs and HSP16.2 in MS illustrate differences between brain and spinal cord in health and upon demyelination. Regional differences in HSP expression may reflect differences in astrocyte cytoskeleton composition and influence inflammation, possibly affecting the effectiveness of pharmacological agents. Multiple sclerosis is a chronic inflammatory disease affecting the whole of the CNS yet very few studies detail the pathology of the spinal cord. Here we show regional differences in HSP expression with higher constitutive expression of HSP in the spinal cord. This may reflect differences in astrocyte cytoskeleton composition, influence the degree of inflammation and thus damage. Such pathological differences are key to understanding diseases processes that may affect local effectiveness of pharmacological agents. [ABSTRACT FROM AUTHOR]

The human small heat-shock protein αB-crystallin is an extremely difficult molecule to study, with its inherent structural dynamics posing unique challenges to all biophysical and structural biology techniques. Here we highlight how the polydispersity and quaternary dynamics of αB-crystallin are intrinsically inter-twined, and how this can impact on measurements of the oligomeric distribution. We show that, in spite of these difficulties, considerable understanding of the varied fluctuations αB-crystallin undergoes at equilibrium has emerged in the last few years. By reporting on data obtained from a variety of biophysical techniques, we demonstrate how the αB-crystallin solution ensemble is governed by molecular motions of varying amplitude and time-scales spanning several orders of magnitude. We describe how these diverse measurements are being used to construct an integrated view of the dynamical structure of αB-crystallin, and highlight areas that require further interrogation. With its study motivating the refinement of experimental techniques, and the development of new approaches to combine the hybrid datasets, we conclude that αB-crystallin continues to represent a paradigm for dynamical biology. [ABSTRACT FROM AUTHOR]

αB-crystallin, also called HspB5, is a molecular chaperone able to interact with unfolding proteins. By interacting, it inhibits further unfolding, thereby preventing protein aggregation and allowing ATP-dependent chaperones to refold the proteins. αB-crystallin belongs to the family of small heat-shock proteins (sHsps), which in humans consists of 10 different members. The protein forms large oligomeric complexes, containing up to 40 or more subunits, which in vivo consist of heterooligomeric complexes formed by a mixture of αB-crystallin and other sHsps. αB-crystallin is highly expressed in the lens and to a lesser extent in several other tissues, among which heart, skeletal muscle and brain. αB-crystallin plays a role in several cellular processes, such as signal transduction, protein degradation, stabilization of cytoskeletal structures and apoptosis. Mutations in the αB-crystallin gene can have detrimental effects, leading to pathologies such as cataract and cardiomyopathy. This review describes the biological roles of αB-crystallin, with a special focus on its function in the eye lens, heart muscle and brain. In addition its therapeutic potential is discussed. [ABSTRACT FROM AUTHOR]

Background: The presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. αB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether αB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces αB-crystallin expression in vitro and in this way may confer hypoxic cell survival. Methods: In 38 HNSCC biopsies, the overlap between immunohistochemically stained αB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of αB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger Nacetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of αB-crystallin on cell survival under hypoxic conditions. Results: In all biopsies αB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased αB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, αB-crystallin expression was increased. αB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced αB-crystallin upregulation. Moreover, it was found that decreased αB-crystallin levels reduced cell survival under hypoxic conditions Conclusions: We provide the first evidence that hypoxia stimulates upregulation of αB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of αB-crystallin may lead to prolonged survival of these cells under hypoxic conditions. [ABSTRACT FROM AUTHOR]

Purpose: The recent discoveries in the field of human small heat shock proteins (sHSPs) clearly point to the important roles played by these adenosine triphosphate (ATP)-independent chaperones in the regulation of a large spectrum of vital cellular processes and in pathological diseases. These proteins are therefore considered as very attractive therapeutic targets. Aims: To understand the functions of the stress-inducible members of the sHSP family, HspB1, HspB5 and HspB8, and be able to therapeutically modulate their activities, researchers are faced with the complex oligomerisation and phosphorylation properties of these proteins and with their ability to interact with each other and with specific protein targets. Here, we have integrated, in a functionally orientated way, the up-to-date literature data concerning HspB1, HspB5 and HspB8 protein interactions which reflect their numerous crucial cellular functions. We also present data supporting the idea that specific phospho-oligomeric domains of HspB1 are involved in the interaction with particular client proteins. Conclusions: More information concerning the interactions between client protein targets and sHSPs or the multiple combinatorial chimeric oligomeric complexes formed by different sHSPs are urgently required to elaborate a comprehensive sHSPs protein interactome and propose efficient and pathology-specific therapeutic approaches. [ABSTRACT FROM AUTHOR]

Historically, small heat shock proteins (sHSPs) have been extensively studied in the context of being intracellular molecular chaperones. However, recent studies looking at the role of sHSPs in neurological diseases have demonstrated a near universal upregulation of certain sHSPs in damaged and diseased brains. Initially, it was thought that sHSPs are pathological in these disease states because they are found in the areas of damage. However, transgenic overexpression and exogenous administration of sHSPs in various experimental disease paradigms have shown just the contrary - that sHSPs are protective, not pathological. This review examines sHSPs in neurological diseases and highlights the potential for using these neuroprotective sHSPs as novel therapeutics. It first addresses the endogenous expression of sHSPs in a variety of neurological disorders. Although many studies have examined the expression of sHSPs in neurological diseases, there are no review articles summarizing these data. Furthermore, it focuses on recent studies that have investigated the therapeutic potential of sHSPs for neurological diseases. Finally, it will explain what we think is the function of endogenous sHSPs in neurological diseases. [ABSTRACT FROM AUTHOR]

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal and pathological cells. Here, we have reviewed the role played by HspB1, HspB4 and HspB5 in the context of Cystic Fibrosis (CF), a severe monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR) some of which trigger its misfolding and rapid degradation, particularly the most frequent one, F508del-CFTR. While HspB1 and HspB4 favor the degradation of CFTR mutants, HspB5 and particularly one of its phosphorylated forms positively enhance the transport at the plasma membrane, stability and function of the CFTR mutant. Moreover, HspB5 molecules stimulate the cellular efficiency of currently used CF therapeutic molecules. Different strategies are suggested to modulate the level of expression or the activity of these small heat shock proteins in view of potential in vivo therapeutic approaches. We then conclude with other small heat shock proteins that should be tested or further studied to improve our knowledge of CFTR processing. [ABSTRACT FROM AUTHOR]

Broilers are more vulnerable to high temperatures than mammals due to the feather cover, lack of sweat glands, fast growth and intensive breeding in commercial systems. Thermal stresses affect the function of various organs and change the expression profiles of hundreds of genes in the different tissues of broilers. Thermal manipulation (TM) during embryogenesis can increase heat tolerance in growing broilers. Small heat shock proteins (SHSPs) are a group of HSPs which participate in many cellular functions like response to different stressors. However, their role in the thermotolerance has not been fully elucidated. Ninety fertilized eggs were randomly divided into three groups (30 eggs/group; 10 eggs/replicate). Normal control (NC) eggs were incubated at 37.5 °C throughout the incubation period whereas heat stress (HS) and cold stress (CS) groups were kept at 41 °C and 33 °C from 15 to 17th day of incubation for 3 h each day, respectively. On day 20, samples from the cerebrums were harvested for histopathology and mRNA expression analyses of HSPB1, HSPB5, HSPB8, and HSPB9. There were no significant differences in survivability, defected embryos, hatchability, and body weight among treatments. TM had no major deleterious effects on the cerebral tissue except for mild degeneration in the HS group. HSPB1, HSPB5, HSPB8, and HSPB9 were expressed in the presence and absence of TM. All SHSP genes tested were downregulated in response to TM except for HSPB9 which was upregulated in the HS group. The highest change in gene expression due to TM observed for HSPB1. This study presents a broader understanding of mechanisms underlying response to TM in broilers. The results suggest that HSPB1, HSPB5, HSPB8, and HSPB9 are involved in thermotolerance in broilers and SHSPs could be involved in the gene expression profiling of TM. It may propose the use of nutritional supplements in the poultry industry to modulate SHSPs. • HSPB1, HSPB5, HSPB8, and HSPB9 are all expressed in both normal and thermal manipulated cerebrums. • The expression of investigated SHSPs in the cerebral tissue is responsive to TM at the late-term embryogenesis of chicken. • HSPB1 is the most responsive gene to the thermal manipulation at the late-term embryogenesis of chicken. • Thermal manipulation had no major deleterious effects on the cerebral tissue except for mild degeneration. • The brain may be protected against TM through SHSPs pathways. [ABSTRACT FROM AUTHOR]

Small heat-shock proteins (sHSPs) are ATP-independent molecular chaperones that interact with partially unfolded proteins, preventing their aberrant aggregation, thereby exhibiting a chaperone-like activity. Dynamics of the quaternary structure plays an important role in the chaperone-like activity of sHSPs. However, relationship between the dynamic structure of sHSPs and their chaperone-like activity remains insufficiently characterized. Many factors (temperature, ions, a target protein, crowding etc.) affect the structure and activity of sHSPs. The least studied is an effect of crowding on sHSPs activity. In this work the chaperone-like activity of HSPB5 was quantitatively characterized by dynamic light scattering using two test systems, namely test systems based on heat-induced aggregation of muscle glycogen phosphorylase b (Phb) at 48 °C and dithiothreitol-induced aggregation of α-lactalbumin at 37 °C. Analytical ultracentrifugation was used to control the oligomeric state of HSPB5 and target proteins. The possible anti-aggregation functioning of suboligomeric forms of HSPB5 is discussed. The effect of crowding on HSPB5 anti-aggregation activity was characterized using Phb as a target protein. The duration of the nucleation stage was shown to decrease with simultaneous increase in the relative rate of aggregation of Phb in the presence of HSPB5 under crowded conditions. Crowding may subtly modulate sHSPs activity. [ABSTRACT FROM AUTHOR]

Ubiquitously expressed human small heat shock proteins (sHsps) HspB1, HspB5, HspB6 and HspB8 contain a conserved motif (S/G)RLFD in their N-terminal domain. For each of them, we prepared mutants with a replacement of the conserved R by A (R/A mutants) and a complete deletion of the pentapeptide (Δ mutants) and analyzed their heterooligomerization with other wild-type (WT) human sHsps. We found that WT HspB1 and HspB5 formed heterooligomers with HspB6 only upon heating. In contrast, both HspB1 mutants interacted with WT HspB6 even at low temperature. HspB1/HspB6 heterooligomers revealed a broad size distribution with equimolar ratio suggestive of heterodimers as building blocks, while HspB5/HspB6 heterooligomers had an approximate 2:1 ratio. In contrast, R/A or Δ mutants of HspB6, when mixed with either HspB1 or HspB5, resulted in heterooligomers with a highly variable molar ratio and a decreased HspB6 incorporation. No heterooligomerization of HspB8 or its mutants with either HspB1 or HspB5 could be detected. Finally, R/A or Δ mutations had no effect on heterooligomerization of HspB1 and HspB5 as analyzed by ion exchange chromatography. We conclude that the conserved N-terminal motif plays an important role in heterooligomer formation, as especially pronounced in HspB6 lacking the C-terminal IXI motif. [ABSTRACT FROM AUTHOR]

Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation. • NPSC differentiation alters proteostasis strategies by rewiring chaperone network • High TRiC/CCT in NSPCs promotes proteome solubility and stress resilience • sHSP induced in neural progeny detoxify misfolded proteins by spatial sequestration • Widespread decline in TRiC/CCT levels and NSPC proteostasis in brain aging Differentiation of neural stem cells (NSPCs) rewires cellular chaperone networks, fundamentally changing cellular proteostasis strategies. Chaperonin TRiC/CCT, high in NSPCs, promotes stress resilience and cellular fitness. Differentiation lowers TRiC/CCT and induces sHSPs, which promote misfolded protein sequestration into protective inclusions. Aging attenuates TRiC/CCT and NSPC proteostasis, likely contributing to proteinopathies. [ABSTRACT FROM AUTHOR]