In addition, stimulation of latently infected resting T cells and macrophages with cytokines, PKC activator, or histone deacetylase inhibitors has been found to greatly upregulate preintegration transcription, leading to low-level viral production or even replication from uDNA. Functionally, Nef synthesized from preintegration transcription is biologically active in modulating host immune functions, lowering the threshold of T cell activation, and downregulating surface CD4, CXCR4/CCR5, and HMC receptors. The early Tat activity from preintegration transcription antagonizes repressive minichromatin assembled onto uDNA. The study of HIV preintegration transcription is important to understanding virus-host interaction and antagonism, viral persistence, and the mechanism of integrase drug resistance. The application of unintegrated lentiviral vectors for gene therapy also offers a safety advantage for minimizing retroviral vector-mediated insertional mutagenesis.Retrovirus integration is an obligatory step for the viral life cycle, but large amounts of unintegrated DNA (uDNA) accumulate during retroviral infection. For simple retroviruses, in the absence of integration, viral genomes are epigenetically silenced in host cells. For complex retroviruses such as HIV, preintegration transcription has been found to occur at low levels from a large population of uDNA even in the presence of host epigenetic silencing mechanisms. HIV preintegration transcription has been suggested to be a normal early process of HIV infection that leads to the syntheses of all three classes of viral transcripts: multiply-spliced, singly-spliced, and unspliced genomic RNA; only viral early proteins such as Nef are selectively translated at low levels in blood CD4 T cells and macrophages, the primary targets of HIV. The initiation and persistence of HIV preintegration transcription have been suggested to rely on viral accessory proteins, particularly virion Vpr and  CEM-50) [Like other retroviruses, HIV was believed to replicate through obligatory steps of reverse transcription and subsequent integration of the viral genome; indeed, integration has been shown to be essential for HIV replication in CD4 T cells and macrophages, the primary targets of HIV infection -14. Yet nd Mo-T) , 16 but  CEM-50)  may resu CEM-50) , 18; bot CEM-50) -26. Cert CEM-50) , 28. In  CEM-50) . However CEM-50) .de novo synthesis of the HIV Tat protein in the absence of integration. It has been demonstrated that HIV Tat-independent transactivation of the LTR promoter can be achieved both by stimulation from non-viral factors, such as cytokines and mitogens present in virus preps [Although not able to replicate in most CD4 T cells, a subset of integration mutants, with mutations in the catalytic residues, have been found to activate the LTR-driving reporter in a HeLa-reporter cell line, suggesting possible viral activities derived from unintegrated viruses that can stimulate cellular activities or the LTR promoter , 30, 31.us preps , 33-40 ous preps . Because2IN/D116N (D116N) was also used and was found to produce Nef [nef gene early without integration in blood T cells [nef in resting T cells, in HIV-1 infection of CD3/CD28-activated or transformed T cells, HIV-1 preintegration transcription was found to be more robust and capable of transcribing all three classes of viral transcripts, namely multiply-spliced, singly-spliced, and unspliced [nef gene was measurably translated. Poon et al. have also found that the synthesis of Nef is dependent on HIV virion Vpr in transformed SUP-T1 T lymphoblast cells; Vpr was found to greatly enhance expression from uDNA [Nef is the first HIV accessory protein that has been demonstrated by western blots to be translated prior to integration in HIV infection of its natural target cells, including blood resting and activated CD4 T cells , 14 and nspliced . Yet, asrom uDNA , 43.nef transcripts from both Wt and D116N infection were also found to be completely resistant to the diketo acid integrase inhibitor L-708,906. Thus, the study concluded that preintegration transcription is a normal early process occurring in productive HIV infection, and did not result from possible peculiarities of certain integrase mutants [A kinetic analysis revealed that preintegration transcription was transient in proliferating T cells; the viral transcripts were detectable as early as 6 hours, peaked at 12-24 hours, and then diminished afterwards . Levels  mutants ; uninteg mutants .1) [nef and tat transcripts and decreased levels of the rev transcript [nef are selectively translated [gag-pol transcript [Semi-quantitative analyses of viral transcription in the presence of integrase inhibitor suggested that HIV transcription can be divided into two phases, early transcription (preintegration transcription) and late transcription (postintegration transcription)  . These t1) . Secondlanscript , 13. Fouanslated , 13. ThiThis biphasic pattern of HIV transcription is reminiscent of gene transcription from DNA viruses, in which gene transcription is divided into early and late phases based on viral DNA replication ; early tnef and tat with highly diminished rev, and there was no detectable viral structural protein synthesis. Only the Nef protein was found to be measurably synthesized and to persist in cells [In proliferating T cells, the transient nature of preintegration transcription from uDNA may result from cell division that can lead to the diminishment of uDNA template. Indeed, in non-proliferating, terminally differentiated macrophages, uDNA was found to be stable, persisting in cells for at least 30 days; preintegration transcription was also found to persist for at least 30 days . Additioin cells . The resin cells , 45.+ T lymphocytes [Functionally, Nef synthesized from preintegration transcription has been shown to be biologically active. In HIV infection of resting CD4 T cells, Nef synthesized prior to integration has been shown to lower the threshold of T cell activation, facilitating productive viral replication upon T cell activation . In HIV phocytes , 46, 47.phocytes . Using Rphocytes . It was phocytes .3tat and rev transcripts are detectable in primary resting CD4 T cells, preactivated and transformed T cells, and macrophages in the absence of viral integration [de novo Tat activities produced from preintegration transcription, Aguilar-Cordova et al. pretreated the LTR reporter cell, 1G5 [de novo Tat functions, Meltzer et al. also constructed two unintegrated GFP HIV reporter viruses with or without the tat gene. When cells were similarly infected , the deletion of Tat greatly diminished GFP expression (96% reduction) from uDNA. This result directly demonstrated that de novo Tat activities are produced from uDNA. Importantly, this uDNA-derived Tat (uTat) can functionally transactivate the unintegrated LTR promoter and antagonize host-mediated chromatin silencing of unintegrated HIV genome [In HIV infection, early egration -14, 51. egration . Demonstegration , 31, 45.ell, 1G5 , with reV genome .de novo production of Rev activity, Wu et al. constructed an HIV Rev-dependent indicator CD4 T cell line, Rev-CEM , which strictly requires Rev interaction with RRE (Rev responsive element) to turn on reporter expression [et al. demonstrated and quantified the de novo generation of Rev activities in the Rev-dependent GFP reporter cells that were infected with D116N or with Wt in the presence of the integrase inhibitor 118-D24-24 [et al. [To demonstrate pression , 49. In pression , 53-57. 8-D24-24 . Similar [et al. , 50. The [et al. , 53.4+ T cells is the full-length, linear, unintegrated DNA [The natural course of HIV infection leads to the accumulation of large amounts of uDNA in infected T cells, lymphoid tissues, and the brain -64. Duriated DNA , 64. In ated DNA . In cellated DNA , 61, 65.ated DNA -68.1) [Three species of uDNA exist in infected cells: the full-length linear DNA, the 1-LTR circles, and the 2-LTR circles  . The cir1) , 70 and 1) . The DNA1) , 73 and 1) . In addi1) . The cir1) , 75. The1) -78. Inde1) . The 2-L1) -83, wher1) , 81, 82 1) -86. The 1) -89. On t1) , 91. In 1) , 92.et al. conducted a flow cytometry sorting of Rev-dependent GFP reporter cells infected with D116N, they discovered that the 2-LTR circles were not found exclusively in the GFP+ cells, but were randomly distributed between GFP+ and GFP- cells [The templates for preintegration transcription have not been clearly identified. In an early transfection experiment, all synthetic LTR-containing DNA circles were found to be active when transiently transfected into HeLa cells . NeverthP- cells . In addiP- cells . These sP- cells , 53. 2-LP- cells . In non-P- cells . It is let al. further quantified HIV preintegration transcription of Rev using the Rev-dependent GFP indicator cells, and found that preintegration transcription occurs on a much larger scale than previously thought [versus 7.2%), only low-expression GFP+ dim cells but not high-expression GFP+ bright cells were generated when integration was inhibited (GPF+ bright cells: 7.4% with integration versus 0.2% without integration). These results suggest a clear distinction between episomal uDNA and proviral template in transcribing genes from the LTR, and in responsiveness to Tat and Rev stimulation [Iyer  thought ; the tra thought . Similar thought ; while tmulation , 52. Evemulation -98.5The accumulation of large amounts of uDNA in cells and the body may trigger host antagonism in silencing preintegration transcription . It has et al. also demonstrated that core histones, the histone variant H3.3, and H1 linker histones are all deposited onto extrachromosomal HIV-1 uDNA early after infection with a Vpr-negative virus [et al. have further demonstrated that in the absence of Vpr, there is a high degree of silencing of uDNA in certain T cell lines, such as K562 and Jurkat [Details of host silencing of retrovirus uDNA have begun to emerge with the studies of minichromatin modification. Nucleosomes were found to be quickly assembled onto the HIV-1 uDNA LTR within 4 hours post-infection , 101 andve virus ; in the ve virus . In addid Jurkat . In partDCAF1 ubiquitin ligase to deplete CTIP2, removing it from the complex of heterochromatin-promoting enzymes for silencing HIV LTR [DCAF1 E3 ligase to degrade histone deacetylase SIRT7 [et al. identified SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1; SFL2-dependent recruitment of SMC5/6 complex to HIV uDNA leads to compaction of viral chromatin and loss of active histone marks. Vpr degrades SLF2 via CRL4DCAF1, thereby increasing chromatin accessibility of uDNA for activating gene expression [Virion Vpr has been shown to greatly antagonize the host silencing machinery, restoring gene expression from uDNA , 43, 103 HIV LTR . Vpr canse SIRT7 . Recentlpression . In addipression . Howeverpression .In addition to Vpr, HIV Tat has also been suggested to partially antagonize host chromatin silencing ; in the Similar chromatin silencing of viral uDNA has also been observed in infection by other retroviruses such as MLV . In MLV 6et al. [et al. [Although certain HIV integrase mutants have been shown to replicate in HTLV-transformed T cell lines , 16, sucet al.  and Kant [et al.  reported [et al.  or stimu [et al. ; it was  [et al. , 100, le [et al. , 100.HDACi-independent replication of unintegrated HIV has also been demonstrated in the infection of blood resting CD4 T cells stimulated with common gamma-chain cytokines , a condiin vitro integration-independent HIV replication observed [in vivo. Certainly, integrase inhibitors are used in drug combinations to treat HIV infection and have shown effectiveness in bringing about faster plasma viral load decay when used in combination with reverse transcriptase (RT) inhibitors [Given the observed , a critihibitors -122. Howhibitors ; five ofhibitors . Importahibitors . As suchhibitors , 99, 100hibitors , 100.in vivo. It also remains to be determined whether uDNA could contribute to residual viremia and some degree of HIV replication in vivo, either directly or through complementation with integrated proviral reservoirs. Recent mathematical modeling has suggested that uDNA could contribute to 20% of total viremia in HIV infection [Somewhat consistent with the animal trial , in humanfection .1) [nef and tat to modulate host cells [The natural course of HIV infection leads to the accumulation of large amounts of unintegrated HIV DNA -63, a li1) , 45, 1081) , 132, itst cells , 14, 95.st cells , 28. uDNst cells -14, 45. st cells , 134, prst cells . uDNA cast cells -14, whicst cells . This cost cells , 28, 135For host cells, silencing uDNA in the nucleus may be an innate response for maintaining the transcriptional fidelity of the host. In addition, silencing uDNA may also avoid viral evasion of host immune surveillance, as Nef expressed from uDNA has been shown to downregulate HMC . The molin vivo role of preintegration transcription in viral pathogenesis and persistence needs to be examined. In vitro, preintegration transcription from uDNA can be greatly upregulated by cytokines or histone deacetylase inhibitors, leading to low-level production of replication-competent viruses [The  viruses , 99, 100 viruses . In treaBasic research on HIV preintegration transcription has also stimulated the use of unintegrated lentiviral vectors for gene therapy , 137-141


A key pathological feature of neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) is the accumulation of aggregated and misfolded protein aggregates with limited effective therapeutic agents. TFEB (transcription factor EB), a key regulator of lysosomal biogenesis and autophagy, plays a pivotal role in the degradation of protein aggregates and has thus been regarded as a promising therapeutic target for these NDs. Here, we systematically summarize the molecular mechanisms and function of TFEB regulation. We then discuss the roles of TFEB and autophagy-lysosome pathways in major neurodegenerative diseases including AD and PD. Finally, we illustrate small molecule TFEB activators with protective roles in NDs animal models, which show great potential for being further developed into novel anti-neurodegenerative agents. Overall, targeting TFEB for enhancing lysosomal biogenesis and autophagy may represent a promising opportunity for the discovery of disease-modifying therapeutics for neurodegenerative disorders though more in-depth basic and clinical studies are required in the future. In nThe lysosome is an organelle for degrading and recycling misfolded and dysfunctional proteins, and it fuses with autophagosomes as autolysosomes to degrade sequestered cargos . ActivatThe aim of this review is to summarize the current knowledge of TFEB-mediated lysosomal biogenesis and autophagy in NDs such as AD and PD, providing novel insight into understanding the pathogenesis of neurodegenerative diseases and the therapeutic potential of TFEB activators. Here, we provide an updated comprehensive understanding of the molecular mechanisms of TFEB activation and its roles in regulating lysosomal biogenesis and autophagy, then discuss its association with NDs with a particular focus on AD and PD, the two most common types of neurodegenerations. Finally, we illustrate several current small molecular TFEB activators and highlight therapeutics potential for targeting TFEB in NDs.2.2.1.LAMP1, CTSD, CTSL, UVRAG, SQSTM1, MAPLC3B, ATG9 and others as shown in Normally, TFEB is inactive and located in the cytoplasm . Upon ac2.2.Legionella effector, acts as a glucosyltransferase that modifies TFEB at multiple sites, including Ser138, Ser195, Ser196, Thr201, Ser203, and Thr208. In particular, its glucosyltransferase activity leads to the impairment of TFEB nuclear export by glucosylation at its Ser138 site, thereby promoting TFEB nuclear accumulation . TF. TFBecn1utophagy , 79 Thisutophagy .Overall, ALP plays a key role in promoting the degradation of α-synuclein, a key pathological protein in PD. Interestingly, neuroinflammation also plays key roles in PD pathogenesis and progression. Since ALP has also been implicated in regulating inflammation, apart from autophagy in neuron cells, future studies for dissection of the crosstalk between neuronal cells and glia cells, and how glia autophagy is regulated in PD will provide novel insight into PD pathogenesis.4.As depicted above, impairment of lysosomal functions and autophagy has been linked to the progression of AD and PD , 81. Her4.1.1-42 and phosphorylated tau levels and subsequent amelioration of cognitive deficits in AD murine models [As aforementioned, the key characteristics of AD are plaques formed by Aβ aggregates and neurofibrillary tangles, which are compromised hyperphosphorylated tau aggregation, accompanied by loss of synapses and neuron death , 82. GSKe models , 84, 85 e models -88. Apare models , 89, 90 APOE4 (apolipoprotein E4) is a major risk gene for AD [APOE4 mutation have a risk of more than 50% for the onset of AD [APOE4 in the modification of TFEB activities. TFEB signaling in AD is summarized in e for AD , 91. Peoet of AD , 93. It et of AD   is increased in PD patients and PD animal models, pharmacological inhibition of c-Abl inhibits GSK3β activity, and thus activates TFEB-mediated lysosomal biogenesis to promote α-synuclein degradation and alleviates PD pathology . Apart fll death .In addition, poly (ADP-ribose) polymerase 1 (PARP1), an enzyme associated with DNA damage, was reported to be activated in PD animal models. PARP1 induces the formation of poly (ADP-ribose) polymers (PAR) and exacerbates the toxicity of α-synuclein . Inhibit4.3.TFEB nuclear translocation is critical for autophagy and lysosomal biogenesis. Hence, discovering drugs that induce TFEB nuclear translocation hold promising for potential therapies for NDs. As aforementioned, TFEB is inactivated via phosphorylation , and thuRecently, multiple small molecules that activate TFEB have been identified. For example, curcumin, CHIR-99021, GSK3β inhibitor VIII and SB216763 induce TFEB nuclear translocation via inhibiting GSK3β , 101. Ac2+-dependent calcineurin activation leads to TFEB dephosphorylation and activation. A variety of small molecules activate TFEB via a calcium-dependent calcineurin pathway. For instance, ML-SA5, a mucolipin 1 (ML1) agonist, induces ML1-mediated Ca2+ release from lysosomes [2+-mediated pathway [2+ levels [2+ release from mitochondrial, leading to TFEB nuclear translocation [2+ storage. Activation of ER-mediated Ca2+ release by exendin-4, ikarugamycin and SB202190 was reported to induce TFEB dephosphorylation and activation [As one of the upstream pathways of TFEB, Caysosomes . In addi pathway , 119. Al+ levels , sulforalocation . The endtivation , 122. OvInterestingly, some small molecule TFEB activators are effective in promoting the degradation of toxic proteins and alleviating the pathologies in multiple NDs, including AD, and PD , 129. Mu5.Caenorhabditis elegans (C. elegans), can also regulate lysosomal function and autophagy [C. elegans and alleviate metabolic diseases by upregulating autophagic genes [C. elegans [C. elegans [Aging is a process of physiologically chronic functional decline, and it is a leading factor in the pathology of many NDs , includiutophagy , 157. Prutophagy . HLH-30/ic genes . Germlin elegans , and HLH elegans . In addi elegans . Three T elegans . As a re6.The hallmarks of NDs including AD and PD are aggregated and misfolded proteins, which compromise normal TFEB functions and its involved lysosomal functions and autophagy. Additionally, several AD and PD-associated risk genes also affect TFEB-mediated ALP. Dysregulated lysosomal functions and autophagy have been reported to be closely relevant to the onset and progression of many NDs such as AD and PD . Thus, iHowever, several questions are still to be clarified in the future. For instance, the specificity of TFEB activators is remained to be elucidated as most of the small molecules have off-targets effects. Targeting Nrf2 signaling also show promising effects in treating NDs including AD and PD. Since recent study showed that TFEB activated Nrf2  and the


However, the cause–effect relationship between STIM1 and glucose/FA metabolism and the possible mechanisms by which STIM1 is involved in these cardiac metabolic diseases are poorly understood. In this review, we first discussed STIM1-dependent signaling in cardiomyocytes and metabolic changes in cardiac hypertrophy and diabetic cardiomyopathy. Second, we provided examples of the involvement of STIM1 in energy metabolism to discuss the emerging role of STIM1 in the regulation of energy substrate preference in metabolic cardiac diseases and speculated the corresponding underlying molecular mechanisms of the crosstalk between STIM1 and cardiac energy substrate preference. Finally, we briefly discussed and presented future perspectives on the possibility of targeting STIM1 to rescue cardiac metabolic diseases. Taken together, STIM1 emerges as a key player in regulating cardiac energy substrate preference, and revealing the underlying molecular mechanisms by which STIM1 mediates cardiac energy metabolism could be helpful to find novel targets to prevent or treat cardiac metabolic diseases.The heart requires a variety of energy substrates to maintain proper contractile function. Glucose and long-chain fatty acids (FA) are the major cardiac metabolic substrates under physiological conditions. Upon stress, a shift of cardiac substrate preference toward either glucose or FA is associated with cardiac diseases. For example, in pressure-overloaded hypertrophic hearts, there is a long-lasting substrate shift toward glucose, while in hearts with diabetic cardiomyopathy, the fuel is switched toward FA. Stromal interaction molecule 1 (STIM1), a well-established calcium (Ca To maintain an optimal contractile function, the heart has a high demand for energy substrates to continuously form energy-rich phosphate bonds  . Multipl2+ sensors. There are two known members of STIM in mammalian cells: STIM1 and STIM2. STIM1 is the major mediator of the classic store-operated calcium entry (SOCE), while STIM2 works more as a coordinator and regulator of STIM1 [2+ concentrations [2+ store depletion, STIM1 and STIM2 become activated and engage with the pore-forming Orai1 ion channel proteins, localized in the plasma membrane, thereby triggering channel opening and activating the previously characterized ICRAC (Ca2+ release activated currents) [2+ imaging techniques to report STIM-Orai1 activity, this process is usually termed Store-Operated Calcium Entry (SOCE) [2+ and STIM1 activation. Future investigations are needed to elucidate this.Stromal interaction molecule (STIM) proteins are endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) Caof STIM1 ,10,11,12trations . Sensingurrents) . As mosty (SOCE) ,9,10. Thy (SOCE) , and cury (SOCE) ,21,22,23y (SOCE) ,25,26. Iy (SOCE) . In addiy (SOCE) . Under ty (SOCE) ,21. Of ny (SOCE) . Interesy (SOCE) ,31  ,34,35,36y (SOCE) , especiaIn this review, we aimed to summarize the currently known roles of STIM1 in the energy metabolism of many types of cells with a focus on cardiomyocytes. We also gave some future perspectives of the possibility of targeting STIM1 to rescue cardiac metabolic diseases.2+-sensing role. There are three coiled-coil regions: coiled coil 1, 2, and 3 . CC1 has an inhibitory effect on CC2–CC3, which is the minimum domain of STIM1 to activate Orai1  [2+ binds to the EF-SAM domain to keep this ER-luminal region monomeric; CC1 interacts with SOAR to maintain cytosolic STIM1 domains in an auto-inhibitory dimeric folding form [2+ store is depleted, the decalcified EF-SAM domains dimerize to pull the TM domains closer, triggering CC1 to release SOAR and unfold the cytosolic region of STIM1, promoting its oligomerization and the activation of Orai1, allowing Ca2+ influxes, or SOCE [STIM1 is a single transmembrane protein resident in ER/SR. It consists of a signal peptide (SP), the EF-hand and sterile alpha motif (EF-SAM) domain in the N-terminus (in the lumen of ER/SR), coiled-coil regions, an inhibitory domain (ID) and a polybasic region (K) in the C-terminus (in cytosol) a. The EFn, SOAR) ,38,39,40ing form ,42,43. W or SOCE   a. STIM1Aof ICRAC . The C-tof ICRAC ,49. STIMof ICRAC .2+ channel Cav2.1 at the T-tubule (2+ ATPase (SERCA) and inositol-1,4,5-triphosphate receptor (IP3R) are also involved in Ca2+ handling in this area  [2+ homeostasis.STIM1-mediated SOCE is crucial for refilling ER Ca2+ ,55,56  . STIM1-kse (UPR) . Single-se (UPR) ,63. Sincse (UPR) ,66,67,682+, STIM1 could also activate downstream Ca2+-dependent metabolic pathways  4 and FA transporter CD36  to the sarcolemma to induce cardiac glucose and FA uptake, respectively [2+ signaling as well [2+ signaling. Moreover, STIM1 can directly interact with a variety of other proteins [In addition to refilling SR Capathways . It is wgulators ,70. For tors [2+ ,72,73. Iectively ,75. More as well ,77. Consproteins ,82,83,84proteins . Interesproteins . Of noteproteins . Therefoproteins  where ST2+ homeostasis or fission. Mitochondrial function including ATP production and reactive oxygen species (ROS) generation is regulated by Ca2+ [2+ via voltage-dependent anion channel (VDAC) and mitochondrial Ca2+ uniporter (MCU) [2+ to mitochondria to regulate mitochondrial function [2+ uptake from the ER/SR [2+ homeostasis due to the changes of STIM1-mediated SOCE would affect ER/SR–mitochondrial Ca2+ communication and thereby cause mitochondrial dysfunction. Excessive or insufficient mitochondrial Ca2+ in cardiomyocytes leads to mitochondrial dysfunction [2+ uptake in diabetic hearts and restoring mitochondrial Ca2+ rescued mitochondrial and cardiac dysfunction [2+–calcineurin is involved in the regulation of mitochondrial fission, which is associated with the development of both cardiac hypertrophy and diabetic cardiomyopathy [2+ level and mitochondrial fission, resulting in the alterations in cardiac glucose and FA oxidation. In turn, increased ROS due to mitochondrial dysfunction could downregulate STIM1 expression through NF-kB as a feedback effect [Moreover, STIM1-mediated SOCE could contribute to cardiac metabolic diseases via the alteration of mitochondrial Ca by Ca2+ ,89. Mitoer (MCU) ,91,92,93function . Therefohe ER/SR ,96,97,98function , which ifunction ,101,102.function . In addimyopathy ,109,110.myopathy ,59. Thismyopathy ,109,110.myopathy , indicatk effect . Moreovek effect . It woulDuring the development of cardiac hypertrophy, there is a shift of energy substrate preference toward glucose utilization, which is caused by the altered expression and activity of transcriptional proteins related to glycolysis ,115,116.In diabetic cardiomyopathy, the balance between glucose and FA utilization shifts to the FA side. As a crucial step, CD36 permanently relocates to the sarcolemma, which increases FA uptake . The inc2+–calcineurin pathway or regulating the expression of GLUT [2+-mediated AMPK phosphorylation at Thr172 [There is a massive number of findings relating STIM1 to glucose metabolism in different organs or cell types. In non-excitable cells, research showed that STIM1-mediated SOCE contributes to glucose uptake via the insulin pathway, Ca of GLUT ,127,128. of GLUT . Likewis of GLUT . Both ef of GLUT . Wilson  of GLUT , and Qiu of GLUT . Differe of GLUT , and the of GLUT , revealit Thr172 . Interest Thr172 , and Wilt Thr172 . Althougt Thr172 , which iKono et al. described a specific reduction in STIM1 expression level in type 2-diabetic islets where insulin-induced glucose metabolism was impaired . SabouriNevertheless, Edwards et al. reported that STIM1 expression and SOCE were increased in coronary smooth muscle with metabolic syndrome, which showed impaired glucose tolerance and insulin resistance . Similar2+ binding affinity, and mutations at the N-glycosylation sites could profoundly affect SOCE [2+-dependent inactivation of STIM1–Orai1 channels [Although it is not so clear if excess glucose would induce glycosylation, some reports showed that increased glucose uptake seems associated with increased N-glycosylation ,151. Intect SOCE ,153. In ect SOCE ,154. Pyrchannels . Taken tThe increased expression of STIM1 has been reported in cardiac hypertrophy ,19,21,22Reports show that STIM1 may promote glucose uptake via its actions on several pathways. STIM1 has been shown to positively modulate the expression levels of GLUT1, GLUT2, and GLUT3 in several types of cells ,130,135.Moreover, STIM1 deletion in cardiomyocytes decreased insulin-induced AKT phosphorylation at Ser473 and increased AS160 phosphorylation which is crucial for insulin-induced glucose uptake ,158. In In addition, AMPK phosphorylation at Thr172, which is important for glucose uptake, is markedly decreased in hearts from cardiomyocyte-restricted STIM1–KO mice . ConsequNevertheless, it is difficult to distinguish if these changes of the key proteins for glucose utilization in diabetic cardiomyopathy are from a direct effect of STIM1 deficiency or a secondary effect of lipid accumulation caused by STIM1 deficiency. Further investigations are needed to provide more information.Consistent with reports showing that STIM1 and STIM1-mediated SOCE are positively involved in glycolysis in several different cells ,129,130,2+-sensitive PDH phosphatase [Glucose oxidation is decreased in diabetic cardiomyopathy and uncoupled with increased glycolysis in cardiac hypertrophy . A recensphatase , and it sphatase ,166. PDKsphatase . Similarsphatase , PDH actsphatase ,119,120.Given that there is an increase in glucose uptake and glycolysis in cardiac hypertrophy , glucose3 production, Ca2+ release from ER, and the consequent activation of STIM1-mediated SOCE in mouse gustatory cells, pancreatic β-cells and mouse podocytes [Researchers showed that the binding of long-chain fatty acids  to CD36 (the major FA transporter in cardiomyocytes ) inducesodocytes ,173,174.odocytes ,176. Howodocytes  and inhiodocytes . In a coodocytes . In geneodocytes . Taken tDrosophila [Khan’s lab found that STIM1-deficient mice lost the spontaneous gustatory preference for fat . Conversosophila . Most fiosophila , which pBased on the aforementioned evidence of the interplay between STIM1 and FA metabolism, we speculate that STIM1 may mediate the FA preference in diabetic cardiomyopathy and that there is an association between STIM1 and altered FA metabolism in cardiac hypertrophy.+-ATPase (v-ATPase) could also enhance CD36 relocation to the sarcolemma in diabetic cardiomyocytes. Given that AMPK phosphorylation at Thr172 is markedly reduced in hearts from cardiomyocyte-restricted STIM1–KO mice [FA uptake into cardiomyocytes is mainly mediated by CD36, and increased FA uptake in diabetic cardiomyopathy is due to a permanent relocation of CD36 to the sarcolemma . Phospho–KO mice , it seem–KO mice ,160. TheAdditionally, a recent report showed that glycogen synthase kinase-3α (GSK-3α) mediates the upregulation of transcription of genes related to fatty acid uptake and storage through PPARα phosphorylation at Ser280 in high-fat diet-induced lipotoxic cardiomyopathy , which sIn terms of how STIM1 is associated with FA oxidation, our speculation is that STIM1 could decrease carnitine-palmitoyl transferase 1 (CPT1) expression or activity, leading to the reduction in FA oxidation in cardiac hypertrophy ➂. CPT1 pIn addition to its inhibition of CPT1 expression, STIM1 also could decrease CPT1 activity in the hearts from cardiomyocyte-restricted STIM1-KO mice . This efPPAR and PGC-1α, which regulate the expression of genes encoding FA oxidation enzymes, were upregulated in diabetic cardiomyopathy, contributing to enhanced FA oxidation . MeanwhiLipid accumulation, which is due to an imbalance between FA uptake and FA oxidation, is one of the key pathologic features of diabetic cardiomyopathy that precedes the onset of contractile dysfunction ,199,200.How increased FA influences STIM1 expression in diabetic cardiomyopathy still needs to be determined. Based on observations from Wilson et al. , a reasoIn this review, we collected evidence to show and discuss the emerging role of STIM1 in the regulation of cardiac energy substrate preference in cardiac metabolic diseases and speculated the possible underlying mechanisms. In cardiac hypertrophy, increased STIM1 expression could induce the energy substrate preference toward glucose through regulating glucose transporters, key kinases and enzymes of energy metabolism and mitochondrial function. Conversely, in diabetic cardiomyopathy, decreased STIM1 levels could induce FA preference by regulating FA transporters, key kinases and enzymes of energy metabolism and mitochondrial function in a way opposite to cardiac hypertrophy. In conclusion, it seems that STIM1 could work as a “decision-maker” of cardiac energy substrate preference.Supporting our speculations, a growing number of studies have shown the role of STIM1 as a potential target to rescue cardiac metabolic diseases. For example, the suppression of STIM1-medaited SOCE, whatever the methods applied , all alleviated the development of cardiac hypertrophy ,207,208.v1.2, SARAF, EB1, Gelsolin, STING, POST, phospholamban, SERCAs, and STIMATE [STIM1 has been reported to interact with a number of proteins including TRPC, Ca STIMATE ,83,84,85 STIMATE . Patient STIMATE . It woulIn summary, it is tempting to see that the crosstalk between STIM1 and energy substrate preference in cardiac metabolic diseases is very complex. The precise causal relationship between them still awaits further elucidation. And this review may provide some possible directions for future studies to explore the precise mechanisms by which STIM1 works as a “balancer” to regulate cardiac energy metabolism.


Recent discoveries shed light on molecular mechanisms responsible for classical Hodgkin lymphoma (HL) development and progression, along with features of Hodgkin – Reed and Sternberg cells (HRS). Here, we summarize current knowledge on characteristic molecular alterations in HL, as well as existing targeted therapies and potential novel treatments for this disease. We discuss the importance of cluster of differentiation molecule 30 (CD30) and the programmed cell death-1 protein (PD-1) and ligands (PD-L1/2), and other molecules involved in immune modulation in HL. We highlight emerging evidence indicating that the altered function of SWI/SNF-type chromatin remodeling complexes, PRC2, and other epigenetic modifiers, contribute to variations in chromatin status, which are typical for HL. We postulate that despite of the existence of plentiful molecular data, the understanding of HL development remains incomplete. We therefore propose research directions involving analysis of reverse signaling in the PD-1/PD-L1 mechanism, chromatin remodeling, and epigenetics-related alterations, in order to identify HL features at the molecular level. Such attempts may lead to the identification of new molecular targets, and thus will likely substantially contribute to the future development of more effective targeted therapies. HL is characterized by CD30 and PD-L1 overexpression, as well as various epigenetic alterations;Further epigenetic study may broaden the knowledge of regulatory processes that are impaired in HL;CD30 and reverse signaling of PD-L1 partially overlap and are interdependent, although the exact molecular mechanism of their interaction remains elusive. Targeting both CD30 and PD-L1 likely represents a more robust strategy to treat HL than existing individual approaches;The precise influence of CD30 and reverse signaling of PD-L1 pathways on chromatin remodeling machinery and epigenetic changes in HL needs to be elucidated;The use of compounds targeting epigenetic machinery aberrant in HL represents an attractive future direction in designing new HL treatments.https://seer.cancer.gov/statfacts/html/hodg.html). HL has a bimodal distribution - it mostly affects young adults, as well as those aged over 50. It is characterized by high cure rates. However, around 10–15% of early stage, and up to 30% patients of advanced stages, experience progressive or relapsed disease after chemotherapy or combined modality treatment. Salvage therapies followed by autologous stem cell transplantation (ASCT) remain the standard therapeutic strategy in the relapse setting, providing long term survival for about 40% of patients. Given that the average age of HL patients is mid-30s, survivors are expected to be economically active in society. Therefore, developing novel therapies, increasing effectiveness of conventional induction strategies, or creating new treatment options for relapse and refractory HL are economically and societally attractive aims.Hodgkin Lymphoma (HL) is rare B-cell lymphoid malignancy with incidence of 2-3 new cases per 100,000 individuals annually. According to NCI NIH database it is estimated that in 2022 the 8,540 new HL cases will be diagnosed and 920 patients will die . These usually represent a few percent of tumor tissue mass, and are surrounded by an abundant polyclonal immune background. The cell type was first observed by Carl Sternberg in 1898, and Dorothy Reed Mendenhall in 1902, in microscope images of lymph nodes . They arRecent discoveries in HRS cells suggest that CD30 or programmed cell death protein-1 receptor and ligands (PD-1/-PD-L1/2) are important pathways for HL development. As such, they have become targets for novel therapeutic strategies , 8. MoreHere, we provide an overview of current knowledge of HL molecular features, targeted therapies and molecular mechanisms. We propose new directions of study aimed at better understanding HL etiology and growth mechanisms. We highlight the significance of reverse signaling in the PD-1/PD-L1 pathway, the role of CD30 signaling, and interplay between these pathways, chromatin-related machinery and epigenetic changes. Collectively, this understanding forms a valuable basis for personalized HL treatment.The survival and proliferation of HRS cells depend on various molecular aberrations, among them signaling involving CD30, and the PD-1/PD-L1,2 pathway seem to be very important.CD30 gene is located on chromosome 1p36  , and theome 1p36 . CD30 isome 1p36 . In diseome 1p36 .via its extracellular domain. After stimulation, receptor trimerization is promoted, enabling signal transduction. The intracellular segment of CD30 then connects with TNFR-associated factors , granzyme B and perforin, as well as leading to deranged cytotoxicity. CD30 suppresses  process . On the  process . Some da process .via internalization of the antibody in the CD30-receptor endocytosis process, resulting in death of the tumor cell  therapy. In clinical trials, CD30 CAR-T therapy was shown to be safe and effective in heavily pre-treated patients with relapsed or refractory HL .PDCD1 gene located on chromosome 2q37.3) is the most studied inhibitory checkpoint  and PD-L2 (CD273 or B7-DC). The genes encoding the two ligands of PD-1, PD-L1 and PD-L2, are located on chromosome 9p24.1, only 42 kilobases apart  of PD-1 receptor, which restores effector T-cell functions, has led to development of novel therapeutic strategies in many tumor types, including HL .Indeed, in HL, blockading the PD-1/PD-L1 axis is beneficial during patient treatment –39. PD-Lloci, as well as by nonmalignant microenvironmental leukocytes  as well as raising the response rate in patients with relapse/refractory HL , 43. Theukocytes . Severalukocytes . A previukocytes .loci encoding PD-L1/2 are located also contains the gene for Janus kinase 2 (JAK2), situated nearly 322 kilobases upstream from PD-L1. Amplification of the entire region increases the cis-influence of the modulation of JAK2 on PD-L1 promoter activity. It consequently enhances JAK2/STAT signaling in HRS cells, leading to both activation and, eventually, suppression , regardless of 9p24.1 copy number amplification (Considering the role of Epstein-Barr virus (EBV) in HL pathogenesis, infection with EBV is one possible inducer of PD-L1 and PD-L2 expression in this condition . EBV profication . These mfication .CD274 gene, which encodes PD-L1, has an AP-1 dependent enhancer that regulates PD-L1 expression in HRS cells. The AP-1 signaling components JunB/c-Jun persistently interact with this enhancer  . Reversevice versa. PD-L1 reverse signaling may lead to the activation of some genes, but whether the CD30-encoding gene is the target remains elusive. Therefore, understanding to what extent the CD30 and PD-L1 pathways overlap is vital, as combined therapy against both molecules may be more effective in refractory and relapse HL  was found to be expressed in the HL microenvironment. Currently, inhibitors of LAG-3 molecule are in clinical trials. The recent data demonstrated that HL resistant for anti-PD1 immunotherapy was depleted in CD8+ T lymphocytes in tumor microenvironment and overexpressed the LAG-3 on CD4+ T lymphocytes. The LAG-3 molecule expression was found on a majority of TILs in pediatric HL . The safAmong various alterations identified in malignant cells, genetic (e.g. mutations) and epigenetic changes play profound roles in cancer development and progression. Epigenetic mechanisms encompass methylation of cytosine residues in DNA, post-translational modification of histone proteins, nucleosome remodeling executed by multi-protein complexes, and actions of regulatory non-coding RNAs. Epigenetic regulatory mechanisms usually influence gene expression at the level of transcription, however they may also affect other steps of gene expression, such as translation. Given its broad regulatory effect on various processes, epigenetics was recently classified as an important hallmark of carcinogenesis. As various epigenetic alterations were reported recently in HL cells , 67, it loci. This modification is introduced by DNA methyltransferases (DNMTs), which transfer the methyl group from the S-adenosyl-L-methionine (SAM) universal donor to the cytosines present in DNA. Therefore, DNA methyltransferases are classified as writers of epigenetic modifications. DNA methylation plays an important role in the control of gene expression at the transcriptional level. In tumorigenesis, CpG islands of the tumor suppressor genes are methylated, frequently resulting in altered chromatin structure, or in direct repression of gene expression. Hyperactivity of DNMTs and alterations in DNA methylation patterns are closely associated with various cancer types. It has been demonstrated that the HL cells exhibit downregulation/silencing of some characteristic genes including CD19, CD20, CD79B, SYK, PU.1, BOB.1/OBF.1, BCMA, and LCK  of specifically silenced genes identified as hypermethylated in HL contained low CpG density in their promoter regions according to the current classification  at the molecular level, thus may be the reason for the appearance of characteristic features of Reed-Sternberg cells  are enzymes that introduce (write) histone acetylation by depositing the acetyl moiety on lysine residues of histone proteins. Such modifications lead to chromatin de-condensation, and subsequently promote gene expression. HATs are categorized into five families with 17 members . In HL, HL cells .Histone deacetylases (HDACs) are enzymes which remove acetyl groups from histone tails, leading to an increased positive charge, therefore promoting histone-DNA interaction. As a consequence, the chromatin structure is more tightly compacted. Thus, their function is the opposite of HATs. HDACs are classified as erasers of histone modifications. They may also act on numerous non-histone proteins such as HSP90, HIF1, and p53 that govern a wide range of biological processes including cancer initiation and progression . The HDACD19, CD20, CD79b, BOB1, PU.1, SYK, LCK, TCL1A, BCMA, PAX5). In contrast, no enrichment for H3K27me3 was found on promoter regions of CCR7, TRAF1, SEMA4C, IL6 genes, which are known to be expressed at high levels in HL. Consistently, the presence of repressive H3K27me3 trimethylation on B-cell characteristic genes correlated with H3 hypoacetylation in HL cells  or protein arginine methyltransferases (PRMTs), which are classified as epigenetic writers. They use S-adenosyl-L-methionine (SAM) as the methyl group donor. Up to three methyl groups may be transferred to the same lysine, and up to two methyl groups may be added on arginine. Histone methylation may positively or negatively impact the expression of target genes. Knowledge regarding the importance of histone methylation in HL development and progression is still very limited. However, there are emerging data indicating that HL cells are characterized by increased levels of histone 3 trimethylation at lysine 27 (H3K27me3), representing a repressive histone mark on the promoter regions of genes involved in the B-cell characteristics . Nftac1 silencing showed correlation with decreased histone H3 acetylation and H3K4 trimethylation, as well as reduced Sp1 factor binding. Increased Heterochromatin Protein 1 (HP1) binding to the f cancer . Furtherpression .Histone demethylation executed by the histone demethylases (erasers) may also play an important role in HL development. It has been reported that KDM6B (JMJD3), the H3K27me3 demethylase, is overexpressed in HL after Epstein-Barr virus (EBV) induction. Depletion of KDM6B in HL cells leads to the increased H3K27 trimethylation on genes differentially expressed in this type of cancer, however the restoration of H3K27me3 did not cause their downregulation . OverexpChromatin remodeling complexes (CRCs) are a group of multisubunit complexes, which are evolutionarily conserved in eukaryotes. CRCs use energy derived from ATP hydrolysis, and act in various ways on nucleosomes. This results in their repositioning, ejection, or exchange, thus giving or restricting access for transcription factors to their target sequences in genomic DNA. CRCs therefore play an important role in the transcriptional control of gene expression , 103.ARID1A (non-core AT-rich interactive domain 1A) gene encoding the BAF250A subunit of SWI/SNF CRC were reported in about 26% of HL patient samples, analyzed by the whole-exome sequencing of flow cytometry–sorted HRS cells from 23 excisional biopsies. The ARID1A acts as a tumor suppressor in multiple solid tumors, and in certain hematologic malignancies such as follicular lymphoma. Of note, the HLs with a ARID1A mutation exhibited an increased number of driver events, compared to HL carrying wild-type ARID1A , two SWI3-type (BAF155 and BAF170), and one SNF5-type (hSNF5/INI1/BAF47) proteins, however non-canonical variants of such complexes reportedly also exist in the cell . Genetice ARID1A . HoweverIn the case of NuRD CRCs, CHD1 core subunit downregulation was a result of promoter methylation . FurtherPcGs represent an important group of proteins that regulate gene expression and chromatin state . Human PCdkn2a) gene plays an important role in cell cycle regulation. It encodes, among others, two proteins known as p16 and p19, which are major inhibitors of cell cycle progression. Cdkn2a is one of the targets of BMI1. A lack of Bmi1 leads to down-regulation of p16 gene in transgenic mice, while 27 of 52 BMI1 positive cases of HL exhibited strong nuclear expression of p16 , which are < 200 bp, and long ncRNAs (lncRNAs), which are >200 bp. sncRNAs are further classified into PIWI-interacting RNAs (piRNAs) or microRNAs (miRNAs), which differ in size and some other properties. ncRNAs can act as modulators in epigenetic or post-transcriptional gene regulation (PTGS). ncRNAs have emerged as both key diagnostic biomarkers and therapeutic targets in various malignancies .It has been shown that the PIWI regulatory pathway may be affected in HL . This paIL15 gene, and the genes HOMER1, SERPINH1 and SUB1, which influence cell growth and survival. mir-148a also plays an important role in negative regulation of proliferation in the KM-H2 HL cell line  is an evolutionarily conserved lncRNA with an 8.7 kb long transcript, encoded on chromosome 11q13. MALAT1 plays a critical role in the maintenance of the proliferation potential of early-stage hematopoietic cells (via mA RNA modification of ZEB1 gene (IDH2) mutations and in murine models of Ten Eleven Translocation 2 (TET2) deficiency. It has been also shown that overexpression of MALAT1 involves hematopoietic stem cell and progenitor cell expression with enhanced inflammation in AML  in HL cell lines, when compared to the control GC-B cell line. 74% of differentially expressed lncRNAs were downregulated in HL. Three lncRNAs  which are upregulated in HL were further analyzed with respect to their function. The inverse expression between LINC00461 and MEF2C (myocyte-specific enhancer factor 2C) suggests an additional potentially important feature of HL , looping chromatin interactions at 3q28, 6q23.3, 10p14 and 16p13.13 regions were found. Subsequently, the biological relevance of these looping interactions was evaluated in HL development. These interactions involved BCL6 and mir-28 at 3q28, MYB and ALDH8A1 at 6q23.3. Regions interacting with TAF3 were found at 10p14. In addition, the region 16p13.13 was found to interact with RMI2 .Epigenetic alterations appear to be of critical importance for HL development Table 2.Alterations in B cell development result in inhibition of tumor suppressor genes expression, leading to transformation into HRS cells. These alterations are driven by multiple mechanisms, including such epigenetic alterations as DNA hypermethylation, enhanced histone deacetylation, histone hypermethylation, missing subunits of chromatin remodeling complexes-CRCs (SWI/SNF and CHD complex), PcG overexpression, increased expression of ncRNAs , dynamic changes in miRNAs profile, and last but not least overexpression of PD-L1 and CD30 molecules  Table 1.vice versa. Chen and coworkers recently indicated the high efficacy of anti- PD-1 treatment after failure of anti- PD-L1 therapy in relapsed/refractory HL, although the patient group in this study was of limited size (Several treatments targeting epigenetic machinery have been recently tested for HL. These include HDAC inhibitors, such as Panobinostat, Mocetinostat or Belinostat , 157. HDted size . Moreoveted size . Study oWriting—original draft preparation, HS, MA, RB, JK, JS, ES, TS, RK. Writing—review and editing, ES, TS, RK, SM, JW, EP-K. Supervision, TS, RK. All authors contributed to the article and approved the submitted version.


Many inborn errors of immunity (IEI) manifest with hallmarks of both immunodeficiency and immune dysregulation due to uncontrolled immune responses and impaired immune homeostasis. A subgroup of these disorders frequently presents with autoimmunity and lymphoproliferation (ALPID phenotype). After the initial description of the genetic basis of autoimmune lymphoproliferative syndrome (ALPS) more than 20 years ago, progress in genetics has helped to identify many more genetic conditions underlying this ALPID phenotype. Among these, the majority is caused by a group of autosomal-dominant conditions including CTLA-4 haploinsufficiency, STAT3 gain-of-function disease, activated PI3 kinase syndrome, and NF-κB1 haploinsufficiency. Even within a defined genetic condition, ALPID patients may present with staggering clinical heterogeneity, which makes diagnosis and management a challenge. In this review, we discuss the pathophysiology, clinical presentation, approaches to diagnosis, and conventional as well as targeted therapy of the most common ALPID conditions. Lymphoproliferation and autoimmunity are relevant manifestations of immuno-hematological diseases. While lymphadenopathy or autoimmune cytopenia in isolation are relatively common and, in most cases, due to secondary causes, the combination of these manifestations raises suspicion for a genetic disease, in particular if associated with other warning signs of an inborn error of immunity (IEI) . These wFAS, FASLG, CASP10) was the first inherited disease presenting with autoimmunity and lymphoproliferation (autoimmune lymphoproliferative immunodeficiency (ALPID) phenotype) that could be linked to a genetic deficiency [Immune responses are highly dynamic and require tight regulation of proliferation and cell death to maintain homeostasis. Impaired immune homeostasis leads to uncontrolled proliferation of immune cells, manifesting as benign or malignant lymphoproliferation in the form of lymphadenopathy, proliferation of mucosa-associated lymphoid tissue, and hepato- or splenomegaly. Uncontrolled, overactive adaptive immune response may also lead to autoimmunity of almost any organ, and favor immune exhaustion or senescence, paradoxically leading to increased infection susceptibility. It is therefore no surprise that many IEI manifest with hallmarks of both immunodeficiency and autoimmune-lymphoproliferative manifestations . Autoimmficiency , 4. Howeficiency , 6, signficiency , activatficiency –11, and ficiency . Patientficiency . Among tficiency . DespiteIn this review, we discuss the pathophysiological basis of the more frequent and mostly autosomal-dominant ALPID conditions and their phenotypic spectrum, as well as laboratory and immunological abnormalities, the approach to diagnosis, and targeted therapies.The development of a functionally competent adaptive immune system involves the generation of a wide repertoire of B (BCR) and T cell receptors (TCR) during the maturation of B and T cells in the bone marrow and the thymus, respectively. A high percentage of TCRs and BCRs generated on maturing cells has been shown to be self-reactive by recognizing self-antigens. In the bone marrow, for instance, up to 75% of the antibodies generated by immature B cells have been reported to be self-reactive . DependiTo maintain peripheral immunological tolerance and prevent autoimmunity, the processes of anergy and apoptosis play an essential role. The two-step process of B and T cell activation is tightly regulated. Major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs) are recognized by TCR on naïve T cells , which in turn may lead to the formation of an immunological synapse favoring engagement of the co-stimulatory or cytokine receptors   . If thisLymphocyte apoptosis contributes to peripheral immune homeostasis by regulating the pool size of certain lymphocyte populations in lymph nodes and spleen. Receptor-mediated apoptosis via the Fas signaling pathway is essential to control unique populations of mammalian targets of rapamycin (mTOR)-dependent hyperproliferative T and B cells, which contain autoreactive specificities. Fas  is a trimeric receptor of the tumor necrosis factor-receptor (TNF-R) family found on a variety of cells including mature lymphocytes. Upon interaction with its ligand, FasL, the Fas receptor recruits the adaptor Fas-associated death domain (FADD) –26. FADDFAS, FASLG, FADD, CASP10) lead to ALPS  [FAS mutations underlie around 15% of all ALPS cases [Mutations in the genes encoding modules of the Fas-FasL apoptotic pathway (henotype  and the henotype . HeterozLPS-FAS) , 4 or soLPS-FAS) , 32. IsoFAS lead to the development of ALPS [In contrast, heterozygous mutations completely abolishing Fas expression, mostly affecting the extracellular or transmembrane domain, do not act dominant-negative, but cause a 50% reduction of total protein expression . These m of ALPS .FAS mutation present at a young age  with lymphadenopathy, splenomegaly, and autoimmunity, mainly but not limited to autoimmune cytopenia [FAS mutation does not necessarily lead to clinical manifestations, even if such individuals exhibit reduced apoptosis, an increased percentage of DNT, and elevated biomarkers such as sFasL and vitamin B12 [Most patients with ALPS due to a ytopenia . Patientamin B12 . A sometamin B12 . Fas-conamin B12 . sFASL aamin B12 –40. FAS-amin B12 . The abeamin B12 . In contamin B12 .FAS ligand deficiency (ALPS-FASLG) follows an autosomal-recessive inheritance and presents clinically like homozygous ALPS-FAS [FASLG mutations has so far been described [CASP8 mutations cause a more complex combined immunodeficiency. Finally, CASP10 mutations were initially reported to be associated with ALPS, but the evidence is not convincing so far [Only a few patients with disease-causing mutations in other components of the Fas pathway have been described in the literature. ALPS-FAS –46. No cescribed –49. FADDescribed –52. Thisg so far , 54.Malignancies, mostly B and T cell lymphomas, and rarely non-lymphoid malignancies have been described in patients with ALPS-FAS with median onset in late adolescence or early adulthood , 55, 56.Inhibitory receptors such as CTLA-4 play an important role in immune regulation and peripheral immunological tolerance by inhibiting immune cell activation . CTLA-4 CTLA4 gene in humans were first described in 2014 , a severe immunodeficiency due to dominant-negative mutations in the AT3 gene , 79. Somof STAT3 , 81. In of STAT3 –84.Fig. STAT3 GOF mutations and clustered them in three groups depending on their molecular activation mechanisms, which correlated to some extent with the observed variable clinical penetrance [STAT3 GOF and STAT5B loss-of-function (LOF)-associated disease [Patients present with variable symptoms, such as lymphoproliferation/splenomegaly, autoimmune cytopenia, type I diabetes in infancy, and enteropathy, as well as short stature and increased susceptibility to viral and bacterial infections , 82, 83.netrance . Some ofnetrance . The def disease .STAT3 GOF mutations lead to reduced Treg populations, in line with the known suppressive effect of STAT3 activity on FoxP3 expression and Treg development [STAT3 mutations, but most frequently, the mutant allele is expressed in a STAT3-deficient cell line followed by a luciferase reporter assay to measure its transcriptional activity [The immune phenotype is not sufficiently characteristic for disease diagnosis. Hypogammaglobulinemia with reduced switched memory and increased CD21low B cells is a common feature, whereas reduced naive CD4 T cells, elevated CD57 + CD8, and DNT cells can be observed in some of STAT3 GOF patients , 84, 89.elopment , 91. Decelopment , 92. Theactivity .STAT3 mutations: in a cohort of 191 patients, 12 developed cancer, most commonly marginal zone B cell and LGL lymphomas [In contrast to other disorders of the ALPID spectrum, malignancy seems to occur less frequently in patients with activating germline ymphomas . STAT3 Gymphomas , 85. Earymphomas . NF-κB transcription factors play an essential role as regulators of the innate and adaptive immunity. The NF-κB family consists of NF-κB1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p100), RelA (p65), c-Rel, and RelB –95. ThesNFKB1 associated with reduced protein levels of the p105 and/or p50 subunit cause a complex immunodeficiency  [NFKB1 mutations, there are no simple screening assays. The p105 and/or p50 levels in transfected cells can be measured via Western blot. Moreover, the mutant allele can be expressed in a cell line and transcriptional activity measured via a NF-κB1-responsive reporter assay [In terms of immune phenotype, most mutation carriers showed reduced switched memory B cells –100. Intaminase) , 104, whaminase) , 101. Inaminase) . Possiblaminase) , decreasaminase) , and incaminase) . RegardiNFKB1 is a tumor suppressor gene, and NF-κB1 haploinsufficiency has been shown to promote tumorigenesis in murine models [e models . Malignae models . Age of e models , 98, wite models . In a coe models .PIK3CD and PIK3R1 genes, respectively [3) by phosphorylating its precursor phosphatidylinositol 4,5-bisphosphate (PIP2). Downstream PIP3 signaling is mediated by intracellular enzymes, such as the serine/threonine kinase AKT, which phosphorylates the FOXO transcription factors inactivating them, as well as regulators of the mTOR complex 1 (mTORC1), which is in turn activated [Class I phosphoinositide 3-kinases (PI3K) play an essential role in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. In the family of class I kinases, PI3Kδ activity is restricted to leukocytes and activated through a variety of receptors, such as cytokine, growth factor, and antigen receptors , 109. PIPIK3CD gene [PIK3R1 gene [PIK3CD E1021K mutation. PIK3R1 encodes the subunits p85α, p55α, and p50α as alternative splicing products; thus, loss-of-function mutations may cause different splice variants and consequently affect PI3Kδ signaling and disease phenotype [Increased activity of the PI3Kδ pathway leads to an autosomal dominant primary immunodeficiency called APDS Fig. . Two ent3CD gene , 9 and A3R1 gene , 11, whi3R1 gene . Thus, mhenotype .Fig. 5IlA hallmark of APDS is recurrent respiratory tract infections as early as in the first year of life in almost all patients, as well as early-onset bronchiectasis  , 112–114Patients often have increased IgM and normal to decreased IgA and total IgG levels, which prior to the first description of APDS often led to the incorrect diagnosis of hyper IgM syndrome (HIGM) . The obsDespite high phenotypic overlap, APDS1 patients more commonly present with bronchiectasis, splenomegaly, cytopenia, and skin disease, whereas APDS2 patients develop growth impairment and lymphoma more frequently . Ten to Most patients with ALPID benefit from conventional PID treatments. Since they often present with hypogammaglobulinemia and/or respond poorly to vaccines, many are treated with immunoglobulin replacement therapy to correct for the secondary antibody deficiency . If recuNext to infection susceptibility, many inborn errors of immunity present with immune dysregulation . The basIn recent years, targeted therapies have become available for several autoimmune-lymphoproliferative diseases. Rapamycin (sirolimus) is an mTOR inhibitor that can be considered a targeted therapy for ALPS patients . mTOR inSince mTOR signaling is activated downstream of PI3Kδ, patients with APDS also benefited from the use of rapamycin, especially in controlling benign lymphoproliferation. However, non-lymphoproliferative complications  are less responsive to mTOR inhibition –114, 138The development of selective PI3Kδ inhibitors, such as leniolisib or idealisib, allows for a specific inhibition of the hyperactive PI3Kδ pathway in APDS patients. A clinical trial of leniolisib (NCT02435173) or seletalisib , oral inhibitors of the p110δ subunit, showed a decrease in lymphoproliferation and some improvement in autoimmune complications (cytopenia) , 140. TrTargeted treatment is also available for patients with CTLA-4 haploinsufficiency or LRBA deficiency. The soluble CTLA-4 immunoglobulin fusion protein abatacept consisting of the Fc-region of human IgG1 linked to the extracellular domain of CTLA-4 can mimic CTLA-4 function. It has been shown to successfully control lymphoproliferation and many autoimmune manifestations, such as cytopenia and enteropathy , 141. InAlthough hyperactive STAT3 cannot be targeted directly, other molecules which are part of the STAT3 pathway can be inhibited. Disruption of the IL-6/IL-6R interaction upstream of STAT3 via the anti-IL-6R monoclonal antibody tocilizumab was shown to partially improve autoimmunity , 85 by iFor ALPID patients, hematopoietic stem cell transplantation (HSCT) is usually restricted to patients with early onset of disease, severe disease manifestations of critical organs , and insufficient response to treatment, including targeted therapies. The hyperactive signaling pathways, resulting in autoimmunity and hyperinflammation, frequently combined with persistent viral infections, poses a great challenge for successful stem cell transplantation . EspeciaThese examples illustrate the clinical and pathophysiological overlaps and differences of selected ALPID diseases. Even within a defined genetic condition, clinical heterogeneity makes diagnosis and management a challenge Table . MoreoveAnother fascinating research topic is the variable clinical penetrance of these mostly autosomal dominant conditions. While some cases may be explained by second-hit somatic mutations leading to clinical manifestation of the disease, other factors that need to be considered are other genetic or epigenetic factors or environmental factors such as microbiota or metabolic cues. A better understanding of penetrance factors may result in prophylactic measures and will allow better discussion of prognosis for affected patients.All figures were created via BioRender.


BRCA1 and BRCA2, many other predisposition genes that confer a moderate risk of BC have been identified. Advances in multigene panel testing have allowed the simultaneous sequencing of BRCA1/2 with these genes in a cost-effective way. Germline DNA from 521 cases with BC fulfilling diagnostic criteria for hereditary BC were screened with multigene NGS testing. Pathogenic (PVs) and likely pathogenic (LPVs) variants in moderate penetrance genes were identified in 15 out of 521 patients (2.9%), including 2 missense, 7 non-sense, 1 indel, and 3 splice variants, as well as two different exon deletions, as follows: ATM (n = 4), CHEK2 (n = 5), PALB2 (n = 2), RAD51C (n = 1), and RAD51D (n = 3). Moreover, the segregation analysis of PVs and LPVs into first-degree relatives allowed the detection of CHEK2 variant carriers diagnosed with in situ melanoma and clear cell renal cell carcinoma (ccRCC), respectively. Extended testing beyond BRCA1/2 identified PVs and LPVs in a further 2.9% of BC patients. In conclusion, panel testing yields more accurate genetic information for appropriate counselling, risk management, and preventive options than assessing BRCA1/2 alone.Breast cancer (BC) is the most common cancer and the leading cause of cancer death in women worldwide. Since the discovery of the highly penetrant susceptibility genes  Breast cancer (BC) is the most common diagnosed cancer and the leading cause of cancer death in women worldwide . A combiBRCA1 and BRCA2 are the genes most commonly associated with hereditary BC, whose proteins are involved in the double-strand DNA damage reparation mechanism . Both these variants were associated with attenuated BC risk  in the RAD51D gene identified in our population study has been previously reported in a BC patient [RAD51C gene, the c.1026+5_1026+7delGTA variant was identified in one patient diagnosed with BC at age 49, without a family history of cancer. Many authors classified this variant as likely pathogenic, as it impacts splicing by causing aberrant exon 8 skipping, resulting in a frame shift and a premature stop codon (p.R322Sfs22*) [RAD51C germline variants by Sanoguera-Miralles et al. (2020) [The contribution of the emerging ,44,45,46. (2020) . They es patient . Concern2Sfs22*) ,51,52,53. (2020) , this alPALB2, CHEK2, ATM, RAD51C, and RAD51D genes. Although there are conflicting points of view in the literature regarding the clinical management of VUS carriers, due to the unknown impact on gene function and cancer risk, the NCCN guidelines recommend screening and risk reduction strategies on the basis of personal and family history [In addition, among all the patients tested, 5.8% had at least one variant of uncertain significance (VUS) identified in the  history ,55,56, wCHEK2 variants c.793-1G>A and p.(Ile157Thr) were found in a ccRCC case and in a subject diagnosed with in situ melanoma, respectively.The segregation analysis, when available, highlighted two major results: CHEK2 alteration and skin cancer (both melanoma and nonmelanoma) is still unclear. Further studies are necessary to explore the involvement of CHEK2 in the development of skin cancer. Nevertheless, CHEK2 is reported to be a key protein in response to UV damage through its upregulation and its phosphorylation by the ATM and ATR proteins [To date, the association between proteins .CHEK2 PVs in a ccRCC patient is not unprecedented. Indeed, a large number of studies have been pointing out a possible connection between CHEK2 alteration and the onset of kidney cancer  [CHEK2 mutation in patients affected by renal cell carcinoma [CHEK2 splicing variant in two individuals who had a diagnosis of ccRCC. Therefore, our finding adds an important piece of evidence to the literature, leading to the need of new more in-depth research to evaluate the role of CHEK2 in renal cancer.The finding of rcinoma) ,60,61,62rcinoma) . Subsequarcinoma ,60,61,62arcinoma  found a RAD51D mutation carriers were more likely to predispose to early-onset BC, with a mean age at diagnosis of 45.4 years, similar to patients with BRCA1 mutation (44.8 years) [RAD51D mutation carriers further confirm this finding.Regarding the clinical and demographic features of the 15 selected patients, the median age of BC onset was about 46 years. This result is approximately in line with previous reports ,17,21,658 years) . The meaMost of the cases (73.3%) exhibited an IDC-NST histology, confirming this as the most common BC histotype .Furthermore, 73% and 53.3% of the cohort showed, respectively, BC and cancer other than BC in their family history. Again, these results are in line with previous studies ,21,65.ATM-mutated BC are characterized by the same HER2, ER, and PR expression levels . This observation further confirms previous studies [RAD51C and RAD51D variants have shown a strong association with triple-negative cancer [RAD51D PV developed two BCs with an opposite receptor profile. Furthermore, a link between the protein-truncating variants in RAD51D and ER-negative breast cancers other than the ER-positive disease has been recently defined [RAD51D, the small size of our population study does not allow for strong evidence. Concerning the CHEK2 gene, the variants have been associated with all BC histotypes except for the triple-negative [CHEK2 PVs carriers did not show a triple-negative molecular status.It is noteworthy that all four subjects displaying an  studies ,69,70 wh studies ,71. For  studies ,71. Alsoe cancer ,70. Here defined . In our negative . Based oGiven the rise in the use of multigene panel testing in diagnostic procedure, the detection of PVs and/or LPVs in a moderate-risk penetrance gene is constantly increasing. In this study, we provide estimates of PV and/or LPV occurrence in genes besides BRCA1/2 and their association either with additional forms of tumors  or with a peculiar molecular signature. Taken all together, these results may help to guide counseling, cancer screening, and other risk management strategies for women and families with PVs and/or LPVs in BC predisposition genes.


All of eight SlSRS proteins were located in the nucleus and possessed transcriptional activity, half of which were transcriptional activators, and the other half were transcriptional repressors. Except for SlSRS1, which showed high transcript accumulation in vegetative organs, most SlSRS genes expressed ubiquitously in all flower organs. In addition, all SlSRS genes could significantly respond to at least four different plant hormones. Further, expression of SlSRS genes were regulated by various abiotic stress conditions. In summary, we systematically analyzed and characterized the SlSRS family, reviewed the expression patterns and preliminarily investigated the protein function, and provided essential information for further functional research of the tomato SRS genes in the determination of reproductive floral organs and the development of plants, and possibly other plants.The SHI RELATED SEQUENCE (SRS) family plays a vital role in the development of multiple plant organs such as floral meristem determinacy, organ morphogenesis, and signal transduction. Nevertheless, there is little understanding of the biological significance of tomato SRS family at this point. Our research identified eight SlSRS family members and classified them into three subfamilies based on phylogenetics, conserved motifs, and characteristic domain analysis. The intraspecies and interspecies collinearity analysis revealed clues of SRS family evolution. Many The online version contains supplementary material available at 10.1186/s12870-023-04506-2. Arabidopsis thaliana [Arabidopsis thaliana mutant shi with gibberellic acid (GA) biosynthesis defect [2CX7CX4CX2C2X6C, where X denotes variable amino acids) which belongs to C3HC3H type ring domain for binding to RNA, protein, and lipid substrates [Arabidopsis SRS protein STYLISH1 (AtSTY1) specifically binds to the ACTCTAC element, in vitro [SRS proteins belong to a plant-specific family of transcription factors contain a cysteine-rich RING-like zinc finger domain and distribute in many plant species and have an important role in the development of organs such as flowers, leaves and roots –6. The fthaliana . Since Fs defect , this fas defect . SRS fambstrates , 2. SRS bstrates . Whetherin vitro . This elSRS genes in Arabidopsis thaliana have been intensively studied. Members of the Arabidopsis thaliana SRS family are functionally conservative and pleiotropic [AtLRP1 expression is affected by auxin and histone deacetylation and it acts downstream of lateral root (LR) forming auxin response modules to negatively regulate LRP development by modulating auxin homeostasis [Arabidopsis thaliana [YUCCA4 (AtYUC4) and AtYUC8 promoters to influence plant development, including cell expansion, stamen and leaf development, and flowering time regulation [At present, SRS TFs have been reported to play a critical role in many physiological and biochemical processes, including hormone biosynthesis, signal transduction, multiple plant organs growth and development, photomorphogenesis, and abiotic stress responses. Some of the 11 iotropic , 9, 10. eostasis , 11, 12.thaliana . AtSTY1 gulation –16. AtSRgulation , promotigulation , and neggulation . AtSRS7 gulation .ZmLRP1 promoter and inhibit its expression [IDEAL PLANT ARCHITECTURE1 (OsIPA1) which acts as a key TF regulating tiller outgrowth and panicle branching [In other species, there are several enlightening researches on the function of SRS TFs. In Populus, two SHI-like proteins have redundant functions on tree growth, form and wood properties . In barlpression . In riceranching .SRS family genes may be involved in abiotic stress tolerance. In the forage species alfalfa (Medicago sativa), expression of MeSRS was induced by cold and salt, suggesting that SRS genes may play important roles in tissue-dependent signaling pathway [Melilotus albus, after undergoing several stresses and hormone treatments, including salinity, low temperature, salicylic acid (SA), and methyl jasmonate (MeJA), expression of MaSRS genes was induced [GmSRS18 of Soybean (Glycine max) has been shown to reduce drought and salt tolerance, as well as stress-related genes expression and physiological indicators such as chlorophyll, proline, and relative electrolyte leakage [Gossypium hirsutum, GhSRS21 negatively regulates salt tolerance in a manner dependent on reactive oxygen species metabolic process [Recently, several studies indicate that  pathway . In Meli induced . GmSRS18 leakage . In Goss process .Solanum lycopersicum L. is an excellent model plant to study fruit development and ripening, quality formation and postharvest preservation technology of fleshy fruits. Expression of Terpenoids 1 (SlEOT1) is the only reported tomato SRS gene [Arabidopsis thaliana AtSTY1. SlEOT1 is specifically expressed in glandular trichomes, and the protein directly activates TERPENE SYNTHASE5 (SlTPS5) transcription and regulates terpene biosynthesis. So far, the SRS TFs have been reported to be involved in the biosynthesis and signal transduction of various phytohormones, suggesting the potential ability to regulate the development of various organs and many physiological and biochemical processes in plants. However, little is known about the molecular mechanism underlying the role of SlSRS TFs in tomato fruit development and quality. Our aim was to investigate SlSRS family’s evolution and function by a systematic comprehensive characterization basedon genome-wide identification, including their phylogenetic relationship, conserved amino acid residues within the RING-like zinc finger domain and IXGH domain, chromosomal distribution and collinearity analysis, and putative cis-elements. Our study also examined their subcellular localization, transcriptional activity, and spatiotemporal expression patterns. Moreover, we detailed the response of SlSRS genes to nine key plant hormones and to various abiotic stresses. Using our results, we provide valuable information about the functional and mechanism analysis of tomato SRS genes. In addition, this study may provide a foundation for future research into plant hormone signaling and stress tolerance.SRS gene , which iSolanum lycopersicum genome were verified by NCBI-CDD. Finally, 8 SlSRS genes were identified in the Solanum lycopersicum genome and named as SlSRS1 to SlSRS8 according to their physical position on tomato chromosomes, among which SlSRS2 was reported and named SlEOT1 [We obtained 8 candidate proteins using HMMER search and 32 candidate proteins using BLASTP tools. After combining results from HMMER search and BLASTP, a total of 32 candidate SlSRS family members in d SlEOT1 . Their bd SlEOT1 . These vArabidopsis thaliana [Glycine max [Oryza sativa [Zea mays, and primitive plants Marchantia polymorpha and Physcomitrella patens, together with Solanum lycopersicum, for phylogenetic analysis. The selected species contain 11, 21, 5, 9, 1, and 2 SRS family members, respectively. As shown in Fig. We selected dicotyledons thaliana  and Glyccine max , monocota sativa  and Zea Arabidopsis thaliana SRS family, we predicted conserved motifs and analyzed characteristic domains of proteins of SlSRS family members using SMART analysis, which is characterized by en enrichment in cysteine residues. Furthermore, they all formed an α-helix at the same position, inside the RING-like zinc finger domain. However, in terms of specific types of amino acid residues, members of Group III were distinctly different from members of Group I and Group II. Looking back at motif analysis, we could find that Motif 1 and Motif 9 both contain the RING-like zinc finger domain, and Motif 2 contains the IXGH domain  and SlSRS8 (Group II) are singleton genes. Subsequently, we calculated the non-synonymous (Ka) and synonymous (Ks) substitution of the collinear SlSRS gene pairs and used their ratios to evaluate the selection pressure during genome evolution. As shown in Table SlSRS genes belonging to Group I. The Ka//Ks ratios between them are all much less than 1, indicating that they have undergone purifying selection during their evolutionary history.Collinearity analysis is helpful to reveal the clues of e 2 Fig. . As showSlSRS genes in other species, collinearity analysis was performed between Solanum lycopersicum and dicotyledons Arabidopsis thaliana and Glycine max, Solanaceous plant Solanum tuberosum, monocots Oryza sativa and Zea mays, and primitive plants Marchantia polymorpha and Physcomitrella patens. It deserves mentioning that primitive plants and tomato do not share any collinear genes, probably due to their distant genetic relationships. Collinear SRS gene pairs were found among tomato, dicotyledons and monocotyledons, but they were all members of Group I. The results of collinearity analysis between tomato and dicotyledons are significantly different to and collinearity analysis between tomato and monocotyledons. In tomato, 30, 38 and 26 collinear gene pairs are found with Arabidopsis thaliana, Glycine max and Solanum tuberosum, respectively, and all six members of Group I have collinear relationships with multiple SRS genes from other species. In contrast, there are only one or two collinear gene pairs among tomato with Oryza sativa and Zea mays. Interestingly, these few collinearity pairs still exist only among members of Group I , and 34 cis-acting elements were identified. Their position and other detailed information were displayed in Fig. SlSRS genes. The other 32 elements could be divided into four categories according to their putative function: light responsive, stress responsive, hormone responsive and developmental regulation. Most of them were considered to be involved in light response, including 3-AF1 binding site, ACE, AE-box, ATCT-motif, Box 4, Box II, chs-CMA1a, circadian, GA-motif, GATA-motif, G-Box, GT1-motif, I-box, LAMP-element, MRE, TCCC-motif, and TCT-motif. In addition, ARE, LTR, MBS, and TC-rich repeats might be involved in responding to drought stress and low temperature stress. The response of auxin, gibberellin, MeJA and other hormones include ABRE, AuxRE, CGTCA-motif, GARE-motif, P-box, TATC-box, TCA-element, TGACG-motif, and TGA-element. Only two cis-acting elements were involved in the developmental regulation of plant tissues and organs: CAT-box, HD-Zip 1.Nicotiana benthamiana) leaf cells to determine the subcellular localization. As shown in Fig. Because nuclear import of transcription factors is instrumental to their transcriptional activity, we investigated the subcellular localization of SlSRS proteins. The SlSRS proteins fused with green fluorescent protein (GFP) was transiently expressed in tobacco  hydrolyzed colorless X-α-Gal to the blue end product and survived from AbA screening, whereas yeast with pGBKT7-SlSRS1, pGBKT7-SlSRS5, pGBKT7-SlSRS7, pGBKT7-SlSRS8 and negative control (empty vector pGBKT7) did not, indicating that SlSRS2, SlSRS3, SlSRS4 and SlSRS6 proteins had transcriptional activation activity. The rest of four SlSRS proteins, SlSRS1, SlSRS5, SlSRS7 and SlSRS8, showed no transcriptional activation activity and were further tested for potential to act as transcriptional repressors using the dual-luciferase system. The full length of coding sequences of these four genes was amplified and cloned into the GAL4BD vector to generate the effectors , flowerorgans at anthesis and 2 days before anthesis  and fruit at different development stages  were detected by qRT-PCR. As shown in Fig. SlSRS1, which shows high transcript accumulation in vegetative organs, most SlSRS genes expressed ubiquitously in all examined flower organs, implying that SlSRS genes likely have important functions in the regulation of tomato flower organs development. The relatively lower expression level of the SlSRS genes was observed in fruits compared to the vegetative organs and the flower organs. However, the expression level of SlSRS5, SlSRS7, and SlSRS8 were gradually increased during fruit ripening, suggesting that they might play a role in fruit ripening. The highest transcript accumulation of SlSRS5 was observed in ovary at anthesis and 2 days before anthesis, suggesting that SlSRS5 might be involved in regulating ovary development or fruit set. Interestingly, SlSRS7 and SlSRS8 showed similar spatial-temporal expression patterns, implying that SlSRS7 and SlSRS8 might have functional redundancy or synergistic effects in regulating tomato plant development.The spatiotemporal expression profiles study contributes to investigating the potential function of the SlSRS genes under different plant hormone treatments could contribute to revealing the potential function of these genes. In this study, we analyzed the responsiveness of SlSRS genes to nine kinds of plant hormones or their analogues by qRT-PCR, including indole-3-acetic acid (IAA), Gibberellin A3 (GA3), 6-Benzylaminopurine (6-BA), ethephon, epi-brassinolide (EBL), salicylic acid (SA), methyl jasmonate (MeJA), Abscisic Acid (ABA) and strigolactone (GR24). Overall, the responsiveness of SlSRS genes to different hormones varied greatly, but all SlSRS genes could respond to at least four kinds of plant hormones. It is noteworthy, SlSRS2, SlSRS5 and SlSRS7 can respond to eight kinds of plant hormones , oxidative (MV), salt (NaCl), droughty (PEG6000), injured (Wound) and osmotic (Mannitol) stresses were assessed by qRT-PCR. In general, the expression of most SlSRS genes could be induced by these six stress treatments. Except for SlSRS2, the other SlSRS genes could respond to at least five kinds of stresses, of which SlSRS1, SlSRS5, SlSRS6, SlSRS7 and SlSRS8 could respond to all six kinds of stresses , which is the structural basis for their transcriptional functions. This is a RING domain rich in acidic Cys and His residues with transcriptional activity [Arabidopsis thaliana, AtLRP1 has been reported to interact with AtSHI, AtSTY1, AtSRS3, AtSRS6 and AtSRS7 proteins of the SRS family [Arabidopsis thaliana AtSRS11, has no IXGH domain that is present in most SRS proteins, suggesting that SlSRS6 has different functions from other members.All SRS TFs contain an extremely conservative RING-like zinc finger domain  in its promoter  and tobacco plants (Nicotiana benthamiana L.) were cultured under the growth conditions of 16/8 h light/dark cycle, 25/20°C day/night temperature and 60% relative humidity. Various organs of tomato plants at different developmental stages were collected in three biological replicates.In this study, tomato plants  PEG6000, 100 mM mannitol, 150 µM methyl viologen (MV) and 200 mM NaCl, respectively, for droughty, osmotic, oxidative and salt stress treatments, and then cultured at standard conditions. Tomato plants with dehydrated stress treatment were removed the soil and cleaned by water, then placed on the filter papers and naturally dried at room temperature. Clean tweezers were used to pierce tomato leaves at the same position for injured stress treatment. The control were well-watered tomato plants. After for 1 h, 3 h, 6 h, 12 and 24 h, leaves at the same position of three individual plants were harvested as one sample. All treated samples were set up in three biological replicates and immediately frozen with liquid nitrogen after collection and stored at −80 °C.https://phytozome-next.jgi.doe.gov/) [Solanum lycopersicum ITAG4.0 [Arabidopsis thaliana TAIR10 [Glycine max Wm82.a4.v1 [Solanum tuberosum v3.0 [Oryza sativa v7.0 [Zea mays Zm-B73-REFERENCE-NAM-5.0 [Marchantia polymorpha v3.1 [Physcomitrella patens v3 [https://www.ebi.ac.uk/interpro/) [http://hmmer.org/) [Arabidopsis and rice SRS protein sequences were used as queries to carry out BLASTP search against tomato proteins. The sequences with E-value < 1e10 were selected for further analysis. Subsequently, all the candidate protein sequences were further examined by Conserved Domain Database (NCBI-CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/) [https://web.expasy.org/protparam/) [Genomic data were obtained from Phytozome (oe.gov/) , includi ITAG4.0 , Arabidoa TAIR10 , Glycine82.a4.v1 , Solanumsum v3.0 , Oryza siva v7.0 , Zea may-NAM-5.0 , Marchanpha v3.1  and Physatens v3 . The hidterpro/) , which wer.org/) . Arabidosb.cgi/) . In additparam/)  was usedhttps://itol.embl.de/index.shtml/) [MUSCLE was used for multiple sequence alignment of the given protein sequences. After being processed by trimAL, the alignment was used to construct the phylogenetic tree by MEGA X  with the.shtml/)  was usedArabidopsis thaliana were used for subsequent analysis. Their motif structures were predicted using Motif-based sequence analysis tools (MEME) web server (https://meme-suite.org/meme/tools/meme) [https://smart.embl.de) [SRS protein sequences from tomato and ls/meme) , using tembl.de)  was usedembl.de)  was usedAdvanced Circos of TBtools  was usedSlSRS genes were obtained by GXF Sequences Extract of TBtools. Next, these sequences were uploaded to the PlantCARE web server (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [The 2000-bp putative promoter regions of the e/html/)  for analSlSRS genes without stop codon was fused into pCXDG-GFP vector. Then the fusion plasmid was transformed into Agrobacterium tumefaciens (GV3101). Transient expression of fusion SlSRS-GFP protein was carried out in leaves of one-month-old tobacco. Three days after infection, the green fluorescence was observed by laser scanning confocal microscope .The full-length coding sequence of SlSRS genes were amplified and ligated into pGBKT7-GAL4BD plasmid which were subsequently transformed into Y2H Gold yeast cells. The yeast was cultured in the SD/Trp medium plate. X-α-gal was used for the identification of the α -galactosidase activity of the transformant, and Aureobasidin A  was used for expression screening.The transcriptional activity of SlSRS proteins were analyzed by the GAL4-responsive reporter system in yeast. The open reading frame (ORFs) of SlSRS1, SlSRS5, SlSRS7 and SlSRS8 were amplified and ligated into pEAQ-GAL4BD plasmid as effector plasmid, with VP16 as a positive control. The double-reporter vector pGreenII 0800-LUC was used as the reporter. The renilla luciferase (REN) driven by CaMV35S was used as the internal control. Agrobacterium tumefaciens (GV3101) with the effector and reporter respectively were co-infected the one-month-old tobacco leaf with the ratio of effector: reporter = 9:1. After 3 days, the activities of LUC and REN were detected by Dual-Luciferase Reporter Assay System . Each combination contained six biological replicates. Finally, the transcriptional activation activity was evaluated by LUC/REN ratio.The ORFs of −ΔΔCt method.Total RNA was extracted using RNAprep Pure Plant Kit  following the manufacturer’s instructions. The first strand of cDNA was synthesized by PrimeScript™ RT reagent Kit with gDNA Eraser  . TB Green® Premix Ex Taq™ II (Tli RNaseH Plus)  was used to perform qRT-PCR on the CFX96 Touch™ Real-Time PCR Detection System . Two microliter 5-fold diluted cDNA was used in each reaction. Finally, the relative expression was calculated by the 2Below is the link to the electronic supplementary material.Supplementary Material 1