Expanding the horizons of targeted protein degradation:A non-small molecule perspective

Targeted protein degradation(TPD)represented by proteolysis targeting chimeras(PROTACs)marks a significant stride in drug discovery.A plethora of innovative technologies inspired by PROTAC have not only revolutionized the landscape of TPD but have the potential to unlock functionalities beyond degradation.Non-small-molecule-based approaches play an irreplaceable role in this field.A wide variety of agents spanning a broad chemical spectrum,including peptides,nucleic acids,antibodies,and even vaccines,which not only prove instrumental in overcoming the constraints of conventional small molecule entities but also provided rapidly renewing paradigms.Herein we summarize the burgeoning non-small molecule technological platforms inspired by PROTACs,including three major trajectories,to provide insights for the design strategies based on novel paradigms.

Machine Learning Modeling of Protein-intrinsic Features Predicts Tractability of Targeted Protein Degradation

Targeted protein degradation(TPD)has rapidly emerged as a therapeutic modality to eliminate previously undruggable proteins by repurposing the cell’s endogenous protein degradation machinery.However,the susceptibility of proteins for targeting by TPD approaches,termed“degradability”,is largely unknown.Here,we developed a machine learning model,model-free analysis of protein degradability(MAPD),to predict degradability from features intrinsic to protein targets.MAPD shows accurate performance in predicting kinases that are degradable by TPD compounds[with an area under the precision–recall curve(AUPRC)of 0.759 and an area under the receiver operating characteristic curve(AUROC)of 0.775]and is likely generalizable to independent non-kinase proteins.We found five features with statistical significance to achieve optimal prediction,with ubiquitination potential being the most predictive.By structural modeling,we found that E2-accessible ubiquitination sites,but not lysine residues in general,are particularly associated with kinase degradability.Finally,we extended MAPD predictions to the entire proteome to find964 disease-causing proteins(including proteins encoded by 278 cancer genes)that may be tractable to TPD drug development.

Increasing Accumulation Level of Foreign Protein in Transgenic Plants Through Protein Targeting

Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.

Physiological functions of Atg6/Beclin 1： a unique autophagy-related protein

The most striking morphological feature of eukaryotic cells is the presence of various membrane-enclosed compartments. These compartments, including organelles and transient transport intermediates, are not static. Rather, dynamic exchange of proteins and membrane is needed to maintain cellular homeostasis. One of the most dramatic examples of membrane mobilization is seen during the process ofmacroautophagy. Macroautophagy is the primary cellular pathway for degradation of long-lived proteins and organelles. In response to environmental cues, such as starvation or other types of stress, the cell produces a unique membrane structure, the phagophore. The phagophore sequesters cytoplasm as it forms a double-membrane cytosolic vesicle, an autophagosome. Upon completion, the autophagosome fuses with a lysosome or a vacuole in yeast, which delivers hydrolases that break down the inner autophagosome membrane along with its cargo, and the resulting macromolecules are released back into the cytosol for reuse. Autophagy is therefore a recycling process, allowing cells to survive periods of nutrient limitation; however, it has a wider physiological role, participating in development and aging, and also in protection against pathogen invasion, cancer and certain neurodegenerative diseases. In many cases, the role ofautophagy is identified through studies of an autophagy-related protein, Atg6/Beclin 1. This protein is part of a lipid kinase complex, and recent studies suggest that it plays a central role in coordinating the cytoprotective function ofautophagy and in opposing the cellular death process of apoptosis. Here, we summarize our current knowledge ofAtg6/Beclin 1 in different model organisms and its unique function in the cell.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase- B/mechanistic target of rapamycin pathway

BACKGROUND The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin(PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills(XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma(HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHPassociated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIM To confirm the effect of XHP on HCC and the possible mechanisms involved.METHODS The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS). Cellbased experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP(0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay.Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction(RT-qPCR), respectively.Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway.Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTS The following 12 compounds were identified in XHP using high-resolution mass spectrometry:Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose-and timedependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract(0.625mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins(e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSION XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3.Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

Targeting amyloid precursor protein shuttling and processing-long before amyloid beta formation

Targeting early steps in amyloid-beta production：Alzheimer’s disease（AD）has a long history as the＂amyloid deposit＂disorder.Many disorders are now known to be caused by proteinβ-sheet misfolding and aggregation（e.g.,Parkinson’s disease：α-synuclein;Huntington’s disease：Huntingtin;

Bone morphogenetic protein signaling：a promising target for white matter protection in perinatal brain injury

Prematurely born newborns,as well as those born at term,may suffer from several types of brain injury including hypoxic-ischemic injury,intracranial hemorrhage,both intraventricular and parenchymal,and injury that is the consequence of intrauterine growth restriction（IUGR）.Injury of all types can impact the motor and cognitive abilities of survivors.The mechanisms leading to disability are not completely understood.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Targeted Degradation of DNA/RNA Binding Proteins via Covalent Hydrophobic Tagging

Targeted protein degradation(TPD)holds great promise for biological inquiry and therapeutic development.However,it still remains elusive to destruct DNA/RNA binding proteins(DBPs/RBPs)previously deemed undruggable.Herein,we report ligandassisted covalent hydrophobic tagging(LACHT)as a modular strategy for TPD of these difficult-totarget proteins.Guided by a noncovalent protein ligand,LACHT leverages a reactive N-acyl-N-alkyl sulfonamide group to covalently label the protein target with a hydrophobic adamantane,which further engages the cellular quality control mechanism to induce proteolytic degradation.Using a smallmolecule ligand,we demonstrated that LACHT allowed TPD of a DBP,bromodomain-containing protein 4,in human leukemia cells with high efficiency.Mechanistic studies revealed that LACHT-mediated TPD dependent on ligand-directed irreversible tagging and the covalently labeled proteins underwent polyubiquitination before removal through both the proteasome and the lysosome.Furthermore,when an RNA ligand was employed,we showed that LACHT also enabled TPD of an RBP,Lin28a,leading to upregulation of its downstream let-7 miRNA.This study thus provides a generalizable platform to expand the TPD toolbox for biomedical applications.

Hydrophobic tag tethering degrader as a promising paradigm of protein degradation:Past,present and future perspectives

Small molecule inhibitors have dominated the pharmaceutical landscape for a long time as the primary therapeutic paradigm targeting pathogenic proteins.However,their efficacy heavily relies on the amino acid composition and spatial constitution of proteins,rendering them susceptible to drug resistance and failing to target undruggable proteins.In recent years,the advent of targeted protein degradation(TPD)technology has captured substantial attention from both industry and academia.Employing an event-driven mode,TPD offers a novel approach to eliminate pathogenic proteins by promoting their degrada-tion,thus circumventing the limitations associated with traditional small molecule inhibitors.Hydropho-bic tag tethering degrader(HyTTD)technology represents one such TPD approach that is currently in the burgeoning stage.HyTTDs employ endogenous protein degradation systems to induce the degrada-tion of target proteins through the proteasome pathway,which displays significant potential for medical value.In this review,we provide a comprehensive overview of the development history and the reported mechanism of action of HyTTDs.Additionally,we delve into the physiological roles,structure-activity re-lationships,and medical implications of HyTTDs targeting various disease-associated proteins.Moreover,we propose insights into the challenges that necessitate resolution for the successful development of HyTTDs,with the ultimate goal of initiating a new age of clinical treatment leveraging the immense po-tential of HyTTDs.

Artificial Nucleic Acid Tractor-Directed Simultaneous Depletion of Oncogenic Membrane Proteins Without Hijacking Proteolysis-Specific Actuator

Targeted protein degradation(TPD)is an emerging tool for degrading proteins of interest,which affords an attractive modality for cancer therapy.However,the present TPD technologies must engage a proteolysis-specific actuator to initiate degradation of targeted proteins in the proteasome or lysosome.Herein,we report an artificial tractor that can induce endocytosis-mediated protein depletion without hijacking a proteolysis-specific actuator.In this design,bispecific aptamer chimeras(BSACs)are established,which can bridge human epidermal growth factor receptor 2(ErbB-2),an important biomarker in a common important biomarker in cancer,with membrane proteins of interest.Taking advantage of the property of aptamer-induced endocytosis and digestion of ErbB-2,another membrane protein is translocated into the lysosome in a hitchhike-like manner,resulting in lysosomal proteolysis along with ErbB-2.This strategy frees the TPD from the fundamental limitation of proteolysis-specific actuator and allows simultaneous regulation of the quantity and function of two oncogenic receptors in a cell-type-specific manner,expanding the application scope of TPD-based therapeutics.

TPX2 knockdown suppressed hepatocellular carcinoma cell invasion via inactivating AKT signaling and inhibiting MMP2 and MMP9 expression

Objective： Targeting protein for Xenopus kinesin-like protein 2 （TPX2） is a nuclear proliferation-related protein that plays a critical role in the formation of mitotic spindle. High expression of TPX2 has been observed in several types of tumors. However, the role of TPX2 in hepatocellular carcinoma （HCC） remains unclear. Our study aimed to investigate the effect of TPX2 on HCC cell invasion. Methods： The immortalized normal human liver cell line L02 and six HCC cell lines including SMMC- 7721, BEL-7402, Huh-7, HepG2, Hep3B and SKHepl were subjected to qRT-PCR and western blot for TPX2 mRNA and protein, respectively. Furthermore, TPX2 small interfering RNA （siRNA） was used to knock down TPX2 expression in SMMC-7721 and HepG2 cells. Cell proliferation and invasion were determined by MTT and transwell assays. Otherwise, expression of p-AKT, MMP2 and MMP9 were evaluated by western blot in SMMC-7721 cells. Results： The expression of TPX2 in HCC cell lines was markedly higher than that in normal human liver cell line. TPX2 knockdown using a specific TPX2-siP, NA reduced the number of invaded cells and inhibited cell proliferation in SMMC-7721 and HepG2 cells. Furthermore, TPX2 knockdown resulted in inactivation of AKT signaling and down-regulation of MMP2 and MMP9 expression in SMMC-7721 cells. Conclusions： Our study identified that TPX2 might contribute to tumor cell invasion through activating AKT signaling and subsequently increasing MMP2 and MMP9 in HCC.

Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells

AIM: To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro.

Effect of You-Gui Yin on glucocorticoid-induced apoptosis of bone marrow mesenchymal stem cells

Objective:To investigate the effect of You-Gui Yin on glucocorticoid-induced apoptosis of rat bone marrow mesenchymal stem cells and its possible mechanism.Methods:20 SD rats were divided into normal saline group and You-Gui Yin group.Ten rats in each group were given normal saline and You-Gui Yin by gavage for 2 weeks,once daily.After the gavage,the rats were sacrificed by spinal removal,blood was taken from the abdominal aorta and centrifuged to obtain blank serum and medicated serum.The rat bone marrow mesenchymal stem cells were extracted and cultured and administered in groups:blank group(10%blank serum),Model group(10%blank serum+dexamethasone 5×10-5mol/ml),traditional Chinese medicine group(10%medicated serum+dexamethasone 5×10-5mol/ml),control group(10%medicated serum),CCK-8 method was used to detect cell proliferation changes in different groups,ELISA method was used to detect bone alkaline phosphatase levels in each group,Alizarin red staining was used to compare red-stained calcium mineralized nodules in each group,Western Blot Detect the expression of apoptosis-related proteins in BMSCs,and perform molecular docking of the quantitative components to evaluate the binding strength and activity of the target and the active compound.Results:The ratio of absorbance(A450)in each time period of the model group was significantly reduced(P<0.05),and the ratio of absorbance(A450)between the traditional Chinese medicine group and the control group was significantly higher than that of the model group(P<0.05).There was no difference between the control group and the blank group(P>0.05);ELISA method showed that the ALP level of the model group was significantly lower than that of the blank group and the control group(P<0.05),the traditional Chinese medicine group significantly increased the ALP level(P<0.05),there was no significant difference between the control group and the blank group;Observation of the number of calcium mineralized nodules under alizarin red staining microscope suggests that the number of calcium mineralized nodules in the model group is significantly lower than that of each group,and there is no significant difference between the control group and the blank group.Both groups are higher than the model group and traditional Chinese medicine Group;Western Blot indicated that compared with the blank group,the expression levels of proapoptotic proteins Bax and Cleaved-casepase-3 in the model group were significantly increased(P<0.01),and the expression levels of anti-apoptotic protein Bcl-2 were decreased(P<0.01),on the contrary in the traditional Chinese medicine group,the expression of the control group did not change significantly compared with the blank group;the molecular docking results showed that You-Gui Yin mainly regulates the apoptosis of BMSCs through Epicatechin 5,7,3'-trimethyl ether,Delphinin,etc.promote bone formation.Conclusion:You-Gui Yin medicated serum can protect BMSCs from apoptosis induced by GCs and promote BMSCs proliferation and osteogenic differentiation by regulating Bax,Cleaved-caspase3,and Bcl-2.

INTRODUCTION OF THE HIGH THROUGHPUT SCREENING SYSTEM

In this article, we introduce the system of high throughput screening (HTS). Its role in new drug study and current development is described. The relationship between research achievements of genome study and new type screening model of new drugs is emphasized. The personal opinions of current problems about HTS study in China are raised.

Uncovering the potential targets of Viburnum odoratissimum for the treatment of related diseases

Viburnum odoratissimum,a landscape plant,is rich in vibsane-type diterpenes,triterpenes,monoterpenoids,sesquiterpenoids,coumarins,and lignans and so on.In the present work,network analysis was carried out to predict the potential targets of V.odoratissimum and the treatment of related diseases.13 main target proteins were predicted,as well as the diseases which might interact with the compounds from V.odoratissimum.The results showed that vibsane-type diterpenes and triterpenes had the potential to treat neoplasm-related and inflammation-related diseases,and the key target proteins were MDM2,NOS,AR,DNMT1,NR3C1,RARA and PTPN1.Among them,AR,NOS,DNMT1,NR3C1,DNMT1 and PTPN1 were also the crucial targets for triterpenes in the treatment of diabetes mellitus.Besides,ALOX15,PIPN1,TLR9,MIT,DNMT1,RAC1,RTGS2,RARA and ACHE were the potential targets for coumarins and lignan in the treatment of neoplasm-related diseases.Furthermore,ESR1,AR,NOS2,NR3C1 were the primary targets for monoterpenoids and sesquiterpenoids because of their potential neuroprotective effects.It is noteworthy that the study of the potential targets and related diseases can provide new insights for further development of V.odoratissimum.

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

Preprotein Import into Chloroplasts via the Toc and Tic Complexes Is Regulated by Redox Signals in Pisum sativum

The import of nuclear-encoded preproteins is necessary to maintain chloroplast function. The recognition and transfer of most precursor proteins across the chloroplast envelopes are facilitated by two membrane-inserted protein complexes, the translocons of the chloroplast outer and inner envelope （Toc and Tic complexes, respectively）. Several signals have been invoked to regulate the import of preproteins. In our study, we were interested in redox-based import regulation mediated by two signals： regulation based on thiols and on the metabolic NADP＋/NADPH ratio. We sought to identify the proteins participating in the regulation of these transport pathways and to characterize the preprotein subgroups whose import is redox-dependent. Our results provide evidence that the formation and reduction of disulfide bridges in the Toc receptors and Toc translocation channel have a strong influence on import yield of all tested preproteins that depend on the Toc complex for translocation. Furthermore, the metabolic NADP＋/NADPH ratio influences not only the composition of the Tic complex, but also the import efficiency of most, but not all, preproteins tested. Thus, several Tic subcomplexes appear to participate in the translocation of different preprotein subgroups, and the redox-active compo- nents of these complexes likely play a role in regulating transport.

Chloroplast Proteins without Cleavable Transit Peptides： Rare Exceptions or a Major Constituent of the Chloroplast Proteome？

Most chloroplast proteins （cp proteins） are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence （cTP）, and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs （non-canonical cp proteins） have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as -30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein （RFP）-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein （PDII）, and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others （＇potential cp proteins＇） were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies.

In vivo Studies on the Roles of Tic55-Related Proteins in Chloroplast Protein Import in Arabidopsis thaliana

The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.