A small fragmented P protein of respiratory syncytial virus inhibits virus infection by targeting P protein.

Peptide-based inhibitors hold promising potential in the development of antiviral therapy. Here, we investigated the antiviral potential of fragmented viral proteins derived from ribonucleoprotein (RNP) components of the human respiratory syncytial virus (HRSV). Based on a mimicking approach that targets the functional domains of viral proteins, we designed various fragments of nucleoprotein (N), matrix protein M2-1 and phosphoprotein (P) and tested the antiviral activity in an RSV mini-genome system. We found that the fragment comprising residues 130-180 and 212-241 in the C-terminal region of P (81 amino acid length), denoted as P Fr, significantly inhibited the polymerase activity through competitive binding to the full-length P. Further deletion analysis of P Fr suggested that three functional domains in P Fr (oligomerization, L-binding and nucleocapsid binding) are required for maximum inhibitory activity. More importantly, a purified recombinant P Fr displayed significant antiviral activity at low nanomolar range in RSV-infected HEp-2 cells. These results highlight P as an important target for the development of antiviral compounds against RSV and other paramyxoviruses.

Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing.

The discovery of CRISPR/Cas has revolutionized the field of genome editing. CRIPSR/Cas components are part of the bacterial immune system and are able to induce double-strand DNA breaks in the genome, which are resolved by endogenous DNA repair mechanisms. The most relevant of these are the error-prone nonhomologous end joining and homology directed repair pathways. The former can lead to gene knockout by introduction of insertions and deletions at the cut site, while the latter can be used for gene correction based on a provided repair template. In this Account, we focus on the delivery aspects of CRISPR/Cas for therapeutic applications in vivo. Safe and effective delivery of the CRISPR/Cas components into the nucleus of affected cells is essential for therapeutic gene editing. These components can be delivered in several formats, such as pDNA, viral vectors, or ribonuclear complexes. In the ideal case, the delivery system should address the current limitations of CRISPR gene editing, which are (1) lack of targeting specific tissues or cells, (2) the inability to enter cells, (3) activation of the immune system, and (4) off-target events. To circumvent most of these problems, initial therapeutic applications of CRISPR/Cas were performed on cells ex vivo via classical methods (e.g., microinjection or electroporation) and novel methods (e.g., TRIAMF and iTOP). Ideal candidates for such methods are, for example, hematopoietic cells, but not all tissue types are suited for ex vivo manipulation. For direct in vivo application, however, delivery systems are needed that can target the CRISPR/Cas components to specific tissues or cells in the human body, without causing immune activation or causing high frequencies of off-target effects. Viral systems have been used as a first resort to transduce cells in vivo. These systems suffer from problems related to packaging constraints, immunogenicity, and longevity of Cas expression, which favors off-target events. Viral vectors are as such not the best choice for direct in vivo delivery of CRISPR/Cas. Synthetic vectors can deliver nucleic acids as well, without the innate disadvantages of viral vectors. They can be classed into lipid, polymeric, and inorganic particles, all of which have been reported in the literature. The advantage of synthetic systems is that they can deliver the CRISPR/Cas system also as a preformed ribonucleoprotein complex. The transient nature of this approach favors low frequencies of off-target events and minimizes the window of immune activation. Moreover, from a pharmaceutical perspective, synthetic delivery systems are much easier to scale up for clinical use compared to viral vectors and can be chemically functionalized with ligands to obtain target cell specificity. The first preclinical results with lipid nanoparticles delivering CRISPR/Cas either as mRNA or ribonucleoproteins are very promising. The goal is translating these CRISPR/Cas therapeutics to a clinical setting as well. Taken together, these current trends seem to favor the use of sgRNA/Cas ribonucleoprotein complexes delivered in vivo by synthetic particles.

Involvement of LARP7 in Activation of SIRT1 to Inhibit NF-κB Signaling Protects Microglia from Acrylamide-Induced Neuroinflammation.

Acrylamide (AM) is a potent neurotoxin and carcinogen that is mainly formed by the Maillard reaction of asparagine with starch at high temperatures. However, the toxicity mechanism underlying AM has not been investigated from a proteomic perspective, and the regulation of protein expression by AM remains poorly understood. This research was the first to utilize proteomics to explore the mechanism of AM exposure-induced neuroinflammation. Target proteins were obtained by differential protein analysis, functional annotation, and enrichment analysis of proteomics. Then, molecular biology methods, including Western blot, qPCR, and immunofluorescence, were used to verify the results and explore possible mechanisms. We identified 100 key differential metabolites by proteomic analysis, which was involved in the occurrence of various biological functions. Among them, the KEGG pathway enrichment analysis showed that the differential proteins were enriched in the P53 pathway, sulfur metabolism pathway, and ferroptosis. Finally, the differential target protein we locked was LARP7. Molecular biological verification found that AM exposure inhibited the expression of LARP7 and induced the burst of inflammation, while SRT1720 agonist treatment showed no effect on LARP7, but significant changes in inflammatory factors and NF-κB. Taken together, these findings suggested that AM may activate NF-κB to induce neuroinflammation by inhibiting the LARP7-SIRT1 pathway. And our study provided a direction for AM-induced neurotoxicity through proteomics and multiple biological analysis methods.

MKRN1 Ubiquitylates p21 to Protect against Intermittent Hypoxia-Induced Myocardial Apoptosis.

Although chronic intermittent hypoxia- (IH-) induced myocardial apoptosis is an established pathophysiological process resulting in a poor prognosis for patients with obstructive sleep apnea syndrome, its underlying mechanism remains unclear. This study is aimed at exploring the role of makorin ring finger protein 1 (MKRN1) in IH-induced myocardial apoptosis and elucidating its molecular activity. First, the GSE2271 dataset was downloaded from the Gene Expression Omnibus database to identify the differentially expressed genes. Then, an SD rat model of IH, together with rat cardiomyocyte H9C2 and human cardiomyocyte AC16 IH models, was constructed. TUNEL, Western blot, and immunohistochemistry assays were used to detect cell apoptosis. Dihydroethidium staining was conducted to analyze the concentration of reactive oxygen species. In addition, RT-qPCR, Western blot, and immunohistochemistry were performed to measure the expression levels of MKRN1 and p21. The direct interaction between MKRN1 and p21 was determined using coimmunoprecipitation and ubiquitination analysis. MKRN1 expression was found to be downregulated in IH rat myocardial tissues as well as in H9C2 and AC16 cells. Upregulated expression of MKRN1 in H9C2 and AC16 cells alleviated the IH-induced reactive oxygen species production and cell apoptosis. Mechanistically, MKRN1 promoted p21 protein ubiquitination and the proteasome pathway degradation to negatively regulate p21 expression. Thus, MKRN1 regulates p21 ubiquitination to prevent IH-induced myocardial apoptosis.

Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.

An endothelial cell inhibitor was purified from supernatant of an Epstein-Barr virus-immortalized cell line and identified as fragments of calreticulin. The purified recombinant NH2-terminal domain of calreticulin (amino acids 1-180) inhibited the proliferation of endothelial cells, but not cells of other lineages, and suppressed angiogenesis in vivo. We have named this NH2-terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin significantly reduced growth of human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenesis inhibitor that specifically targets proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.

Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa.

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.

Genome Editing in a Wide Area of the Brain Using Dendrimer-Based Ternary Polyplexes of Cas9 Ribonucleoprotein.

A preassembled Cas9/single-guide RNA complex (Cas9 ribonucleoprotein; Cas9 RNP) induces genome editing efficiently, with small off-target effects compared with the conventional techniques, such as plasmid DNA and mRNA systems. However, penetration of Cas9 RNP through the cell membrane is low. In particular, the incorporation of Cas9 RNP into neurons and the brain is challenging. In the present study, we have reported the use of a dendrimer (generation 3; G3)/glucuronylglucosyl-ß-cyclodextrin conjugate (GUG-ß-CDE (G3)) as a carrier of Cas9 RNP and evaluated genome editing activity in the neuron and the brain. A Cas9 RNP ternary complex with GUG-ß-CDE (G3) was prepared by only mixing the components. The resulting complex exhibited higher genome editing activity than the complex with the dendrimer (G3), Lipofectamine 3000 or Lipofectamine CRISPRMAX in SH-SY5Y cells, a human neuroblastoma cell line. In addition, GUG-ß-CDE (G3) enhanced the genome editing activity of Cas9 RNP in the whole mouse brain after a single intraventricular administration. Thus, GUG-ß-CDE (G3) is a useful Cas9 RNP carrier that can induce genome editing in the neuron and brain.

TRIM21 and PHLDA3 negatively regulate the crosstalk between the PI3K/AKT pathway and PPP metabolism.

PI3K/AKT signaling is known to regulate cancer metabolism, but whether metabolic feedback regulates the PI3K/AKT pathway is unclear. Here, we demonstrate the important reciprocal crosstalk between the PI3K/AKT signal and pentose phosphate pathway (PPP) branching metabolic pathways. PI3K/AKT activation stabilizes G6PD, the rate-limiting enzyme of the PPP, by inhibiting the newly identified E3 ligase TIRM21 and promotes the PPP. PPP metabolites, in turn, reinforce AKT activation and further promote cancer metabolic reprogramming by blocking the expression of the AKT inhibitor PHLDA3. Knockout of TRIM21 or PHLDA3 promotes crosstalk and cell proliferation. Importantly, PTEN null human cancer cells and in vivo murine models are sensitive to anti-PPP treatments, suggesting the importance of the PPP in maintaining AKT activation even in the presence of a constitutively activated PI3K pathway. Our study suggests that blockade of this reciprocal crosstalk mechanism may have a therapeutic benefit for cancers with PTEN loss or PI3K/AKT activation.

The La autoantigen contains a dimerization domain that is essential for enhancing translation.

The La autoantigen is an RNA-binding protein that is involved in initiation and termination of RNA polymerase III transcription. It also binds several viral RNAs, including those of poliovirus and human immunodeficiency virus (HIV). Binding of the La protein to these RNAs enhances their translation in vitro (K. Meerovitch, Y.V. Svitkin, H.S. Lee, F. Lejbkowicz, D.J. Kenan, E.K.L. Chan, V.L. Agol, J.D. Keene, and N. Sonenberg, J. Virol. 67:3798-3807, 1993, and Y.V. Svitkin, A. Pause, and N. Sonenberg, J. Virol. 68:7001-7007, 1994). Here, a functional domain in the carboxy-terminal half of La that is distinct from the RNA-binding domain is described. Deletion of this domain abrogated the ability of La protein to enhance translation of poliovirus RNA and a hybrid HIV trans-activation-response element-chloramphenicol acetyltransferase mRNA. Far-Western assays indicated that the La protein homodimerized in vitro, and the C-terminal deletions that caused a loss of activity in translation also abrogated the dimerization signal. Gel filtration chromatography of recombinant La protein confirmed that La protein exists as a dimer under native conditions. Addition of the purified dimerization domain resulted in a loss of translation stimulatory activity of La protein in cell-free-translation reactions.

La autoantigen is required for the internal ribosome entry site-mediated translation of Coxsackievirus B3 RNA.

Translation initiation in Coxsackievirus B3 (CVB3) occurs via ribosome binding to an internal ribosome entry site (IRES) located in the 5'-untranslated region (UTR) of the viral RNA. This unique mechanism of translation initiation requires various trans-acting factors from the host. We show that human La autoantigen (La) binds to the CVB3 5'-UTR and also demonstrate the dose-dependent effect of exogenously added La protein in stimulating CVB3 IRES-mediated translation. The requirement of La for CVB3 IRES mediated translation has been further demonstrated by inhibition of translation as a result of sequestering La and its restoration by exogenous addition of recombinant La protein. The abundance of La protein in various mouse tissue extracts has been probed using anti-La antibody. Pancreatic tissue, a target organ for CVB3 infection, was found to have a large abundance of La protein which was demonstrated to interact with the CVB3 5'-UTR. Furthermore, exogenous addition of pancreas extract to in vitro translation reactions resulted in a dose dependent stimulation of CVB3 IRES-mediated translation. These observations indicate the role of La in CVB3 IRES-mediated translation, and suggest its possible involvement in the efficient translation of the viral RNA in the pancreas.

Transcription efficiency of human polymerase III genes in vitro does not depend on the RNP-forming autoantigen La.

Transcription of class III genes is conducted by multi-protein complexes consisting of polymerase III itself and several transcription factors. We established a reconstituted in vitro transcription system from which the autoantigen La was removed by immunodepletion. This system showed no RNP formation, but was still fully active in transcription. Supplementing such La-free transcription reactions with recombinant La restored the formation of La complexes with the newly synthesised RNA, but did not lead to enhanced transcription efficiency. Furthermore, we developed a technique for the generation and isolation of transcription complexes, assembled from purified transcription factors and isolated by glycerol centrifugation. These complexes were fully competent to re-initiate RNA synthesis but they were not associated with La and their transcription rate could not be stimulated by addition of recombinant La. Therefore, we conclude that La does not act as a human polymerase III transcription factor.

Swine TRIM21 restricts FMDV infection via an intracellular neutralization mechanism.

The tripartite motif protein 21 (TRIM21) is a ubiquitously expressed E3 ubiquitin ligase and an intracellular antibody receptor. TRIM21 mediates antibody-dependent intracellular neutralization (ADIN) in cytosol and provides an intracellular immune response to protect host defense against pathogen infection. In this study, swine TRIM21 (sTRIM21) was cloned and its role in ADIN was investigated. The expression of sTRIM21 is induced by type I interferon in PK-15 cells. sTRIM21 restricts FMDV infection in the presence of FMDV specific antibodies. Furthermore, sTRIM21 interacts with Fc fragment of swine immunoglobulin G (sFc) fused VP1 of FMDV and thereby causing its degradation. Both the RING and SPRY domains are essential for sTRIM21 to degrade sFc-fused VP1. These results suggest that the intracellular neutralization features of FMDV contribute to the antiviral activity of sTRIM21. sTRIM21 provide another intracellular mechanism to inhibit FMDV infection in infected cells.

Nuclear ribonucleoproteins as inhibitors of mammalian RNA polymerase.

A ribonucleoprotein complex isolated from rabbit thymus nuclear lysates was found to be an inhibitor of DNA-dependent RNA polymerase II. The inhibition appeared to be of a competitive type and was completely reversed by high concentration of DNA. Highest inhibition was observed when enzyme and complex were preincubated before addition of DNA while there was little inhibition after enzyme had started synthesis on the DNA template. The RNA isolated from the complex was equally inhibitory and was a more effective inhibitor than either tRNA or rRNA.

Polypyrimidine tract-binding protein inhibits translation of bip mRNA.

Translation initiation of human Bip mRNA is directed by an internal ribosomal entry site (IRES) located in the 5' non-translated region. No trans-acting factor possibly involved in this process has as of yet been identified. For the encephalomyocarditis virus and other picornaviruses, polypyrimidine tract-binding protein (PTB) has been found to enhance the translation through IRES elements, probably by interaction with the IRES structure. Here, we report that PTB specifically binds to the central region (nt 50-117) of the Bip 5' non-translated region. Addition of purified PTB to rabbit reticulocyte lysate and overexpression of PTB in Cos-7 cells selectively inhibited Bip IRES-dependent translation. On the other hand, depletion of endogenous PTB or addition of an RNA interacting with PTB enhanced the translational initiation directed by Bip IRES. These suggest that PTB can either enhance or inhibit IRES-dependent translation depending on mRNAs.

Calreticulin inhibits vitamin D's action on the PTH gene in vitro and may prevent vitamin D's effect in vivo in hypocalcemic rats.

1,25-dihydroxyvitaminD3 [1,25-(OH)2D3] and PTH both act to increase serum calcium. In addition, 1,25-(OH)2D3 decreases PTH gene transcription, which is relevant both to the physiology of calcium homeostasis and to the management of the secondary hyperparathyroidism of patients with chronic renal failure. In chronic hypocalcemia there is secondary hyperparathyroidism with increased levels of PTH mRNA and serum PTH despite markedly increased levels of 1,25-(OH)2D3. We have studied the role of calreticulin in this resistance to 1,25-(OH)2D3. Weanling rats fed a low-calcium diet were hypocalcemic and had increased PTH mRNA levels despite high serum 1,25-(OH)2D3 levels. 1,25-(OH)2D3 given by continuous minipump infusion to normal rats led to the expected decrease in PTH mRNA. The hypocalcemic rats had an increased concentration of calreticulin in the nuclear fraction of their parathyroids, but not in other tissues. Gel shift assays showed that a purified vitamin D receptor and retinoid X receptor-beta bound to the PTH promoter's chicken and rat vitamin D response element (VDRE), and this binding was inhibited by added pure calreticulin. Transfection studies with a PTH VDRE-chloramphenicol acetyltransferase (CAT) construct showed that 1,25-(OH)2D3 decreased CAT transcription. Cotransfection of PTH VDRE-CAT with a calreticulin expression vector in the sense orientation prevented the transcriptional effect of 1,25-(OH)2D3, but a calreticulin vector in the antisense orientation had no effect. These results show that calreticulin prevents the binding of vitamin D receptor-retinoid X receptor-beta to the PTH VDRE in gel retardation assays and prevents the transcriptional effect of 1,25-(OH)2D3 on the PTH gene. This is the first report of calreticulin inhibiting a down-regulatory function of a sterol hormone and may help explain the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to 1,25-(OH)2D3.

Laminin binding to the calreticulin fragment vasostatin regulates endothelial cell function.

Vasostatin, the 1-180 amino acids NH(2) domain of calreticulin, inhibits endothelial cell proliferation, angiogenesis, and tumor growth, but the mechanisms underlying these effects are unclear. We show that endothelial cells express the extracellular matrix protein laminin, including chains alpha 5 and gamma 1 and that vasostatin specifically binds to laminin. When added to endothelial cell cultures, vasostatin specifically inhibits endothelial cell attachment to laminin and by this mechanism, can reduce subsequent endothelial cell growth induced by basic fibroblast growth factor. As an angiogenesis inhibitor that specifically disrupts endothelial cell attachment to components of the extracellular matrix, vasostatin has a unique potential as a cancer therapeutic.

An RNA Hybridization Assay for Screening Influenza A Virus Polymerase Inhibitors Using the Entire Ribonucleoprotein Complex.

Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.

The angiogenesis inhibitor vasostatin does not impair wound healing at tumor-inhibiting doses.

Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair.

Translational control during early development.

The regulation of gene expression during early development is controlled predominantly at the translational level. This becomes necessary due to transcriptional arrest during maturation of the oocyte and the rapid early cleavage divisions of the embryo. Consequently, early events involved in pattern formation, germ cell specification, cell fate, and cell division are programmed by maternal messenger ribonucleic acid (RNA). The majority of these transcripts are stored as masked ribonucleoproteins particles during oogenesis but undergo sequence-specific translational activation during oocyte maturation and embryogenesis. Translational regulation occurs in the male germ line and, though not a prominent feature, in the soma as well. Evidence suggests that the assembly of ribonucleoprotein (RNP) particles may be coupled to transcription. Specific translational regulatory sequences are found in the 5' and 3' untranslated regions of maternal mRNA, but the majority occur downstream of the coding region. Most of the known cis-acting sequences and associated proteins are implicated in translational repression. Antisense RNA also has been implicated in specific mRNA translational regulation. Although little is known about message-specific unmasking, in general, one exception is cytoplasmic polyadenylation-mediated translational activation. A widespread phenomenon, it occurs in both vertebrates and invertebrates.

Calreticulin modulates the in vitro DNA binding but not the in vivo transcriptional activation by peroxisome proliferator-activated receptor/retinoid X receptor heterodimers.

Calreticulin is a ubiquitous calcium binding/storage protein found primarily in the endoplasmic reticulum. Calreticulin has been shown to inhibit DNA binding and transcriptional activation by glucocorticoid and androgen hormone receptors by binding to the conserved sequence KXFF(K/R)R, present in the DNA-binding domains of all known members of the steroid/nuclear hormone receptor superfamily. To determine whether calreticulin might be a general regulator of hormone-responsive pathways, we examined its effect on DNA binding in vitro and transcriptional activation in vivo by heterodimers of the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR alpha). We show here that purified calreticulin inhibits the binding of PPAR/RXR alpha heterodimers and of other nuclear hormone receptors, to peroxisome proliferator-responsive DNA elements in vitro. However, overexpression of calreticulin in transiently transfected cultured cells had little or no effect on transactivation mediated by PPAR/RXR alpha. Therefore, while calreticulin inhibits the binding of both nuclear and steroid hormone receptors to cognate response elements in vitro, our findings suggest that calreticulin does not necessarily play an important role in the regulation of all classes of hormone receptors in vivo.